FOXM1 Contributes to Chemotherapy Sensitivity in Cervical Cancer by Regulating TTK.

BACKGROUND: The emergence of chemotherapy resistance usually causes therapeutic failure in advanced cervical cancer. Forkhead box protein M1 (FOXM1) and threonine tyrosine kinase (TTK) are closely associated with cancer drug sensitivity, but the mechanism of FOXM1 on TTK involvement in chemo-treated cervical cancer remains unclear. Here, we aimed to observe the effects of FOXM1 on TTK and on chemotherapy sensitivity in cervical cancer. METHODS: The expressions of FOXM1 and TTK in cervical cancer tissues and para-cancerous tissues were analyzed by immunohistochemistry. SiHa and Hela cells were transfected with human lentivirus-FOXM1, small interfering RNA (siRNA) or pcDNA3.1/FOXM1 to analyze the changes in TTK protein expression. Furthermore, the cells were treated with paclitaxel (8 µM) or cisplatin (10 µM) to analyze the effects of FOXM1 on chemotherapy sensitivity. SiHa cells were used to construct a xenograft model to study the effects of FOXM1 expression in response to paclitaxel treatment. The tumor size and weight were observed. The expressions of Ki-67, FOXM1, and TTK protein in tumor tissues were measured by immunohistochemistry. RESULTS: High expression of FOXM1 and TTK were found in the cervical cancer tissues (p < 0.05). The TTK protein expressions were decreased by FOMX1-siRNA transfection in SiHa and Hela cells (p < 0.01). The cell viability and cell cycle were also suppressed by FOMX1-siRNA transfection (p < 0.01) but enhanced by pcDNA3.1/FOXM1 transfection (p < 0.01). For paclitaxel or cisplatin treatment, the cell viability and cell DNA damage were improved due to the FOXM1 overexpression (p < 0.01). TTK inhibitor significantly suppressed the effects of FOXM1 overexpression (p < 0.01). CONCLUSIONS: FOXM1 regulated TTK and affected the therapeutic efficacy of cisplatin and paclitaxel in cervical cancer.

High-throughput assessment identifying major platelet Ca2+ entry pathways via tyrosine kinase-linked and G protein-coupled receptors.

In platelets, elevated cytosolic Ca2+ is a crucial second messenger, involved in most functional responses, including shape change, secretion, aggregation and procoagulant activity. The platelet Ca2+ response consists of Ca2+ mobilization from endoplasmic reticulum stores, complemented with store-operated or receptor-operated Ca2+ entry pathways. Several channels can contribute to the Ca2+ entry, but their relative contribution is unclear upon stimulation of ITAM-linked receptors such as glycoprotein VI (GPVI) and G-protein coupled receptors such as the protease-activated receptors (PAR) for thrombin. We employed a 96-well plate high-throughput assay with Fura-2-loaded human platelets to perform parallel [Ca2+]i measurements in the presence of EGTA or CaCl2. Per agonist condition, this resulted in sets of EGTA, CaCl2 and Ca2+ entry ratio curves, defined by six parameters, reflecting different Ca2+ ion fluxes. We report that threshold stimulation of GPVI or PAR, with a variable contribution of secondary mediators, induces a maximal Ca2+ entry ratio of 3-7. Strikingly, in combination with Ca2+-ATPase inhibition by thapsigargin, the maximal Ca2+ entry ratio increased to 400 (GPVI) or 40 (PAR), pointing to a strong receptor-dependent enhancement of store-operated Ca2+ entry. By pharmacological blockage of specific Ca2+ channels in platelets, we found that, regardless of GPVI or PAR stimulation, the Ca2+ entry ratio was strongest affected by inhibition of ORAI1 (2-APB, Synta66) > Na+/Ca2+ exchange (NCE) > P2×1 (only initial). In contrast, inhibition of TRPC6, Piezo1/2 or STIM1 was without effect. Together, these data reveal ORAI1 and NCE as dominating Ca2+ carriers regulating GPVI- and PAR-induced Ca2+ entry in human platelets.

Amuvatinib Blocks SARS-CoV-2 Infection at the Entry Step of the Viral Life Cycle.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). SARS-CoV-2 propagation is mediated by the protein interaction between viral proteins and host cells. Tyrosine kinase has been implicated in viral replication, and hence, it has become a target for developing antiviral drugs. We have previously reported that receptor tyrosine kinase inhibitor blocks the replication of hepatitis C virus (HCV). In the present study, we investigated two receptor tyrosine kinase-specific inhibitors, amuvatinib and imatinib, for their potential antiviral efficacies against SARS-CoV-2. Treatment with either amuvatinib or imatinib displays an effective inhibitory activity against SARS-CoV-2 propagation without an obvious cytopathic effect in Vero E6 cells. Notably, amuvatinib exerts a stronger antiviral activity than imatinib against SARS-CoV-2 infection. Amuvatinib blocks SARS-CoV-2 infection with a 50% effective concentration (EC50) value ranging from ~0.36 to 0.45 µM in Vero E6 cells. We further demonstrate that amuvatinib inhibits SARS-CoV-2 propagation in human lung Calu-3 cells. Using pseudoparticle infection assay, we verify that amuvatinib blocks SARS-CoV-2 at the entry step of the viral life cycle. More specifically, amuvatinib inhibits SARS-CoV-2 infection at the binding-attachment step. Moreover, amuvatinib exhibits highly efficient antiviral activity against emerging SARS-CoV-2 variants. Importantly, we demonstrate that amuvatinib inhibits SARS-CoV-2 infection by blocking ACE2 cleavage. Taken together, our data suggest that amuvatinib may provide a potential therapeutic agent for the treatment of COVID-19. IMPORTANCE Tyrosine kinase has been implicated in viral replication and has become an antiviral drug target. Here, we chose two well-known receptor tyrosine kinase inhibitors, amuvatinib and imatinib, and evaluated their drug potencies against SARS-CoV-2. Surprisingly, amuvatinib displays a stronger antiviral activity than imatinib against SARS-CoV-2. Amuvatinib blocks SARS-CoV-2 infection by inhibiting ACE2 cleavage and the subsequent soluble ACE2 receptor. All these data suggest that amuvatinib may be a potential therapeutic agent in SARS-CoV-2 prevention for those experiencing vaccine breakthroughs.

ELF3 promotes gemcitabine resistance through PKMYT1/CDK1 signaling pathway in gallbladder cancer.

BACKGROUND: Gemcitabine is the standard treatment for gallbladder cancer (GBC) patients, and the development of resistance frequently limits its efficacy. However, the molecular features and mechanisms of gemcitabine resistance (Gem-R) in GBC cells remain unknown. Herein, we aimed to explore the role of ELF3 in Gem-R of GBC, including the underlying mechanisms. METHODS: RNA sequencing was used to screen the essential genes related to the generation of Gem-R in GBC tissues. The correlation between Gem-R and ELF3 expression was identified in GDSC, GEO database, GBC tissues, and 3 GBC cell lines. Immunohistochemical staining, quantitative real-time polymerase chain reaction, and western blot were used to examine the expression of ELF3, PKMYT1, and CDK1. Luciferase reporter assays were used to identify the binding site of ELF3 in the PKMYT1 promoter region. CCK-8 assay and clonogenic survival assays were used to evaluate the sensitivity of gemcitabine in GBC cells. A GBC xenograft model was used to evaluate the influence of ELF3 on the therapeutic effect of gemcitabine. RESULTS: A consistently positive correlation between ELF3 expression and Gem-R, both in newly generated GBC RNA-seq data and in the datasets from GDSC and GEO. Gem-R in GBC cells was facilitated by ELF3 overexpression, whereas ELF3 knockdown had the opposite effect. In vivo experiments further proved that reducing ELF3 expression promoted the gemcitabine sensitivity of GBC cells and extended the survival time of mice that received orthotopic xenografted tumors. Mechanistically, ELF3 upregulated PKMYT1 expression by interacting with the DNA binding region of PKMYT1 in GBC cells, thereby promoting the phosphorylation of CDK1 and inducing Gem-R. Treatment with a combination of the PKMYT1 shRNA and gemcitabine significantly reduced the growth of GBC cells induced by overexpression of ELF3 in vitro and in vivo. CONCLUSIONS: ELF3/PKMYT1/CDK1 axis significantly regulates Gem-R to GBC cells and may represent a promising drug target for treating GBC patients.

(R)-9bMS Inhibited the Protein Synthesis and Autophagy of Triple Negative Breast Cancer Cells via Regulating miR-4660/mTOR Axis.

BACKGROUND: Unlike other subtypes of breast cancer, triple negative breast cancer (TNBC) exhibits aggressive and metastatic behaviors and a lack of effective targeted therapeutics. (R)-9bMS, a small-molecule inhibitor of the non-receptor tyrosine kinase 2 (TNK2), significantly inhibited TNBC cell growth; however, the functional mechanism of (R)-9bMS in TNBC remains largely unknown. OBJECTIVE: To explore the functional mechanism of (R)-9bMS in TNBC. METHODS: Cell proliferation, apoptosis and xenograft tumor growth assays were performed to evaluate the effects of (R)-9bMS on TNBC. The expression levels of miRNA and protein were detected by RTqPCR or western blot, respectively. Protein synthesis was determined by analyzing the polysome profile and 35S-met incorporation. RESULTS: (R)-9bMS attenuated TNBC cell proliferation, induced cell apoptosis, and inhibited xenograft tumor growth. Mechanism study indicated that (R)-9bMS upregulated the expression of miR-4660 in TNBC cells. The expression of miR-4660 is lower in TNBC samples than that of the non-cancerous tissues. miR-4660 overexpression inhibited TNBC cell proliferation by targeting the mammalian target of rapamycin (mTOR), which reduced mTOR abundance in TNBC cells. Consistent with the downregulation of mTOR, exposure of (R)-9bMS inhibited the phosphorylation of p70S6K and 4E-BP1, which consequently interrupted the total protein synthesis and autophagy of TNBC cells. CONCLUSION: These findings uncovered the novel working mechanism of (R)-9bMS in TNBC by attenuating mTOR signaling via up-regulating miR-4660. The potential clinical significance of (R)- 9bMS in TNBC treatment is interesting to explore.

Compound K is a potential clinical anticancer agent in prostate cancer by arresting cell cycle.

BACKGROUND: Ginsenosides, phenolic compounds, and polysaccharides are the bioactive constituents of Panax ginseng Meyer. Compound K (CK) is a secondary ginsenoside with better bioavailability. It is also a promising anticancer agent. PURPOSE: We aimed to evaluate the effect of CK on prostate cancer (PCa) and its potential mechanisms. STUDY DESIGN: The proliferation, migration and cell cycle of PCa cells after CK treatment were assessed in various PCa cell lines. Docetaxel was used as a positive control drug. Unlike other published studies, the potential mechanisms of CK (50 µM) were investigated by an unbiased global transcriptome sequencing in the current study. METHODS: Key CK related genes (CRGs) with prognostic significance were identified and verified by bioinformatic methods using data from the TCGA dataset and GSE21034 dataset. The role of CDK1 in the effect of CK treatment on PCa cells was investigated by overexpression of CDK1. RESULTS: CK inhibited the proliferation and migration of PCa cells at concentrations (less than 25 µM) without obvious cytotoxicity. Five key CRGs with prognostic significance were identified, including CCNA2, CCNB2, CCNE2, CDK1, and PKMYT1, which are involved in cell cycle pathways. CK inhibited the expression of these 5 genes and the cell cycle of PCa cells. According to the results of bioinformatic analysis, the expression of the five key CRGs was strongly associated with poor prognosis and advanced pathological stage and grade of PCa. In addition, CK could restore androgen sensitivity in castration-resistant PCa cells, probably by inhibiting the expression of CDK1. After CDK1 overexpression, the inhibition of proliferation and migration of PCa cells by CK was decreased. The inhibition on the phosphorylation of AKT by CK was also reduced. CONCLUSION: CK can inhibit PCa cells, and the mechanisms may be associated with the inhibition of cell cycle pathways through CDK1. CK is also a potential clinical anticancer agent for treating PCa.

Long-lasting memory of jasmonic acid-dependent immunity requires DNA demethylation and ARGONAUTE1.

Stress can have long-lasting impacts on plants. Here we report the long-term effects of the stress hormone jasmonic acid (JA) on the defence phenotype, transcriptome and DNA methylome of Arabidopsis. Three weeks after transient JA signalling, 5-week-old plants retained induced resistance (IR) against herbivory but showed increased susceptibility to pathogens. Transcriptome analysis revealed long-term priming and/or upregulation of JA-dependent defence genes but repression of ethylene- and salicylic acid-dependent genes. Long-term JA-IR was associated with shifts in glucosinolate composition and required MYC2/3/4 transcription factors, RNA-directed DNA methylation, the DNA demethylase ROS1 and the small RNA (sRNA)-binding protein AGO1. Although methylome analysis did not reveal consistent changes in DNA methylation near MYC2/3/4-controlled genes, JA-treated plants were specifically enriched with hypomethylated ATREP2 transposable elements (TEs). Epigenomic characterization of mutants and transgenic lines revealed that ATREP2 TEs are regulated by RdDM and ROS1 and produce 21 nt sRNAs that bind to nuclear AGO1. Since ATREP2 TEs are enriched with sequences from IR-related defence genes, our results suggest that AGO1-associated sRNAs from hypomethylated ATREP2 TEs trans-regulate long-lasting memory of JA-dependent immunity.

3,6'-Disinapoylsucrose alleviates the amyloid precursor protein and lipopolysaccharide induced cognitive dysfunction through upregulation of the TrkB/BDNF pathway.

The aim of this study is to explore the effect and mechanism of 3,6'-disinapoylsucrose (DISS) on an Alzheimer's disease (AD) mice model induced by APPswe695 lentivirus (LV) and intraperitoneal injection of lipopolysaccharide (LPS). The results show that DISS improves cognitive ability, decreases the levels of IL-2, IL-6, IL-1ß, and TNF-α, reduces the expression of NF-κB p65, and alleviates Aß deposition and nerve cell damage. DISS can regulate tyrosine kinase B (TrkB)/brain-derived neurotrophic factor (BDNF) signaling in the hippocampus. In summary, DISS can significantly alleviate neuroinflammation, spatial learning and memory disorders in AD model mice.

Improving endothelial cell junction integrity by diphenylmethanone derivatives at oxidative stress: A dual-action directly targeting caveolar caveolin-1.

Directly targeting caveolar caveolin-1 is a potential mechanism to regulate endothelial permeability, especially during oxidative stress, but little evidence on the topic limits therapeutics discoveries. In this study, we investigated the pharmacological effect of an antioxidant LM49 (5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanoe) and its five diphenylmethanone derivatives on endothelial permeability and establish two distinct mechanisms of action. Multiplex molecular assays with theoretical modeling indicate that diphenylmethanone molecules, including LM49, directly bind the caveolin-1 steric pocket of ASN53/ARG54, ILE49/ASP50, ILE18, LEU59, ASN60, GLU48 and ARG19 residues. They also indicated dynamic binding-affinity for diphenylmethanone derivatives. First, this molecular interaction at caveolin-1 pocket inhibits its phosphorylation at TYR14 residue in H2O2-injured endothelial cell. A positive correlation was established between diphenylmethanone derivative binding-affinity and caveolin-1 phosphorylation inhibition. Inhibition of caveolin-1 phosphorylation, however, was independent of the LM49-mediated variation of protein tyrosine kinase activity, suggesting a direct blockage of adenosine triphosphate substrate diffusion into cavelion-1 structure. Second, LM49 increases the expression of cellular adhesive and tight junction proteins, VE-cadherin and occludin, in H2O2-injured cell, in a dose dependent manner. A leakage assay of fluorescein isothiocyanate-labeled dextran 40 across cell monolayer suggested improvement in endothelial barrier integrity with diphenylmethanone treatments. Our results demonstrate a direct targeting effect of caveolin-1 on endothelial permeability, and should guide the diphenylmethanone therapy against oxidative stress-induced junction dysfunction, especially at caveolar membrane invagination.

Protein tyrosine kinase inhibitor resistance in malignant tumors: molecular mechanisms and future perspective.

Protein tyrosine kinases (PTKs) are a class of proteins with tyrosine kinase activity that phosphorylate tyrosine residues of critical molecules in signaling pathways. Their basal function is essential for maintaining normal cell growth and differentiation. However, aberrant activation of PTKs caused by various factors can deviate cell function from the expected trajectory to an abnormal growth state, leading to carcinogenesis. Inhibiting the aberrant PTK function could inhibit tumor growth. Therefore, tyrosine kinase inhibitors (TKIs), target-specific inhibitors of PTKs, have been used in treating malignant tumors and play a significant role in targeted therapy of cancer. Currently, drug resistance is the main reason for limiting TKIs efficacy of cancer. The increasing studies indicated that tumor microenvironment, cell death resistance, tumor metabolism, epigenetic modification and abnormal metabolism of TKIs were deeply involved in tumor development and TKI resistance, besides the abnormal activation of PTK-related signaling pathways involved in gene mutations. Accordingly, it is of great significance to study the underlying mechanisms of TKIs resistance and find solutions to reverse TKIs resistance for improving TKIs efficacy of cancer. Herein, we reviewed the drug resistance mechanisms of TKIs and the potential approaches to overcome TKI resistance, aiming to provide a theoretical basis for improving the efficacy of TKIs.

[Combination of RNase Binase and AKT1/2 Kinase Inhibitor Blocks Two Alternative Survival Pathways in Kasumi-1 Cells].

Treatment of malignant neoplasms often requires the use of combinations of chemotherapeutic agents. However, in order to select combinations that are effective against specific tumor cells, it is necessary to understand the mechanisms of action of the drugs that make up the combination. Bacillus pumilus ribonuclease (binase) is considered as an adjuvant antitumor agent, and the sensitivity of malignant cells to the apoptogenic effect of binase depends on the presence of certain oncogenes. In the acute myelogenous leukemia cell line Kasumi-1, binase blocks the proliferation pathway mediated by the mutant tyrosine kinase KIT, which, as shown in our work, activates an alternative proliferation pathway through AKT kinase. In Kasumi-1 cells, binase in combination with an Akt1/2 inhibitor induces apoptosis, and their toxic effects add up: the Akt1/2 inhibitor blocks the binase-induced pathway after suppression of the KIT-dependent pathway. Thus, a combination of binase and AKT kinase inhibitors can effectively block various pathways of tumor cell proliferation and be used for their elimination.

Tyrosine Kinase Inhibition Alters Intratumoral CD8+ T-cell Subtype Composition and Activity.

Targeted therapy with a tyrosine kinase inhibitor (TKI) such as imatinib is effective in treating gastrointestinal stromal tumor (GIST), but it is rarely curative. Despite the presence of a robust immune CD8+ T-cell infiltrate, combining a TKI with immune-checkpoint blockade (ICB) in advanced GIST has achieved only modest effects. To identify limitations imposed by imatinib on the antitumor immune response, we performed bulk RNA sequencing (RNA-seq), single-cell RNA-seq, and flow cytometry to phenotype CD8+ T-cell subsets in a genetically engineered mouse model of GIST. Imatinib reduced the frequency of effector CD8+ T cells and increased the frequency of naïve CD8+ T cells within mouse GIST, which coincided with altered tumor chemokine production, CD8+ T-cell recruitment, and reduced CD8+ T-cell intracellular PI3K signaling. Imatinib also failed to induce intratumoral T-cell receptor (TCR) clonal expansion. Consistent with these findings, human GISTs sensitive to imatinib harbored fewer effector CD8+ T cells but more naïve CD8+ T cells. Combining an IL15 superagonist (IL15SA) with imatinib restored intratumoral effector CD8+ T-cell function and CD8+ T-cell intracellular PI3K signaling, resulting in greater tumor destruction. Combination therapy with IL15SA and ICB resulted in the greatest tumor killing and maintained an effector CD8+ T-cell population in the presence of imatinib. Our findings highlight the impact of oncogene inhibition on intratumoral CD8+ T cells and support the use of agonistic T-cell therapy during TKI and/or ICB administration.

Combinatorial targeting of a chromatin complex comprising Dot1L, menin and the tyrosine kinase BAZ1B reveals a new therapeutic vulnerability of endocrine therapy-resistant breast cancer.

BACKGROUND: Targeting vulnerabilities of cancer cells by inhibiting key regulators of cell proliferation or survival represents a promising way to overcome resistance to current therapies. In breast cancer (BC), resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor alpha (ERα) signaling to the genome. Targeting components of the ERα pathway in these tumors represents, therefore, a rational way toward effective new treatments. Interaction proteomics identified several proteins associated with ERα in BC cells, including epigenetic complexes controlling gene transcription comprising the scaffold protein menin and the histone methyltransferase Dot1L. METHODS: We combined chromatin immunoprecipitation, transcriptome sequencing, siRNA-mediated gene knockdown (kd), pharmacological inhibition coupled to cellular and functional assays and interaction proteomics in antiestrogen (AE)-sensitive and AE-resistant human BC cell models to: map menin and Dot1L chromatin localization, search for their common and specific target genes, measure the effects of single or combinatorial knockdown or pharmacological inhibition of these proteins on cell proliferation and survival, and characterize their nuclear interactomes. RESULTS: Dot1L and menin associate in MCF-7 cells chromatin, where they co-localize in a significant fraction of sites, resulting in co-regulation of genes involved, among others, in estrogen, p53, HIF1α and death receptor signaling, regulation of cell cycle and epithelial-to-mesenchymal transition. Specific inhibitors of the two factors synergize with each other for inhibition of cell proliferation of AE (tamoxifen or fulvestrant)-sensitive and AE-resistant BC cells. Menin and Dot1L interactomes share a sizeable fraction of their nuclear partners, the majority being known BC fitness genes. Interestingly, these include B-WICH and WINAC complexes that share BAZ1B, a bromodomain protein comprising a tyrosine-protein kinase domain playing a central role in chromatin remodeling and transcriptional regulation. BAZ1B kd caused significant inhibition of ERα expression, proliferation and transcriptome changes resulting in inhibition of estrogen, myc, mTOR, PI3K and AKT signaling and metabolic pathways in AE-sensitive and AE-resistant BC cells. CONCLUSIONS: Identification of a functional interplay between ERα, Dot1L, menin and BAZ1B and the significant effects of their co-inhibition on cell proliferation and survival in cell models of endocrine therapy-resistant BC reveal a new therapeutic vulnerability of these aggressive diseases.

Sex-specific endothelial dysfunction induced by high-cholesterol diet in rats: The role of protein tyrosine kinase and nitric oxide.

BACKGROUND AND AIMS: Atherosclerosis is a chronic process playing a crucial role in the pathogenesis of cardiovascular disease. Sex-specific differences in the incidence of atherosclerosis indicate that estrogen has a protective effect on the cardiovascular disease. However, the role of sex on endothelium responses in animal models of high cholesterol (HC) diet-induced atherosclerosis has not been fully investigated. This study was aimed to investigate vascular responses in HC-fed rats. METHODS AND RESULTS: Male and female Sprague rats (12-week-old) were treated with either a standard diet (n = 12 of each sex) or an HC enriched diet (n = 12 of each sex) containing 2% cholesterol for 24 weeks. HC treated animals (both sexes) showed increased levels of total cholesterol, LDL-cholesterol, triglyceride and blood pressure (BP) compared to control rats. While the BP of control rats (both sexes) was increased following aminoguanidine administration (AG, 100 mg/kg i.p.), it was not changed in HC animals (both sexes). The hypotensive effect of acetylcholine was significantly impaired in male HC-treated rats. In vitro experiments demonstrated that aortic rings from HC group (both sexes) had an increased contractile response to phenylephrine and a decreased vasodilatory response to acetylcholine. The vasorelaxant effect of acetylcholine in HC rats (only male) was improved by applying 10-5 M genistein (tyrosine kinase inhibitor) or AG. CONCLUSION: HC diet alters endothelium function through Nitric oxide (NO) and tyrosine kinase pathways in male rats.

Methionine oxidation of CLK4 promotes the metabolic switch and redox homeostasis in esophageal carcinoma via inhibiting MITF selective autophagy.

BACKGROUND: Metabolic reprogramming and redox homeostasis contribute to esophageal squamous cell carcinoma (ESCC). CDC-like kinase 4 (CLK4) is a dual-specificity kinase that can phosphorylate substrates' tyrosine or serine/threonine residue. However, the role and mechanism of CLK4 in ESCC remain unknown. METHODS: CLK4 expression was analysed using publicly available datasets and confirmed in ESCC tissues and cell lines. The biological roles of CLK4 were studied with gain and loss-of-function experiments. Mass spectrometry was employed to examine the effects of CLK4 on metabolic profiling. In vitro kinase assay, co-immunoprecipitation, glutathione S-transferase pulldown, chromatin immunoprecipitation and luciferase reporter were used to elucidate the relationship among CLK4, microphthalmia-associated transcription factor (MITF), COP1 and ZRANB1. RESULTS: CLK4 down-regulation was observed in ESCC cell lines and clinical samples and associated with the methylation of its promoter. Low levels of CLK4 promoted ESCC development by affecting the purine synthesis pathway and nicotinamide adenine dinucleotide phosphate (NADPH)/nicotinamide adenine dinucleotide phosphate (NADP+ ) ratio. Interestingly, CLK4 inhibited ESCC development by blocking MITF-enhanced de novo purine synthesis and redox balance. Mechanistically, wild type CLK4 (WT-CLK4) but not kinase-dead CLK4-K189R mutant phosphorylated MITF at Y360. This modification promoted its interaction with E3 ligase COP1 and its K63-linked ubiquitination at K308/K372, leading to sequestosome 1 recognition and autophagic degradation. However, the deubiquitinase ZRANB1 rescued MITF ubiquitination and degradation. In turn, MITF bound to E- rather than M-boxes in CLK4 promoter and transcriptionally down-regulated its expression in ESCC. Clinically, the negative correlations were observed between CLK4, MITF, and purine metabolic markers, which predicts a poor clinical outcome of ESCC patients. Notably, CLK4 itself was a redox-sensitive kinase, and its methionine oxidation at M307 impaired kinase activity, enhanced mitochondria length and inhibited lipid peroxidation, contributing to ESCC. CONCLUSIONS: Our data highlight the potential role of CLK4 in modulating redox status and nucleotide metabolism, suggesting potential therapeutic targets in ESCC treatment.

Morphine Prevents Ischemia/Reperfusion-Induced Myocardial Mitochondrial Damage by Activating δ-opioid Receptor/EGFR/ROS Pathway.

OBJECTIVE: The purpose of this study was to determine whether the epidermal growth factor receptor (EGFR), which is a classical receptor tyrosine kinase, is involved in the protective effect of morphine against ischemia/reperfusion (I/R)-induced myocardial mitochondrial damage. METHODS: Isolated rats hearts were subjected to global ischemia followed by reperfusion. Cardiac H9c2 cells were exposed to a simulated ischemia solution followed by Tyrode's solution to induce hypoxia/reoxygenation (H/R) injury. Triphenyltetrazolium chloride (TTC) was used to measure infarct size. The mitochondrial morphological and functional changes were determined using transmission election microscopy (TEM), mitochondrial stress assay, and mitochondrial swelling, respectively. Mitochondrial fluorescence indicator JC-1, DCFH-DA, and Mitosox Red were used to determine mitochondrial membrane potential (△Ψm), intracellular reactive oxygen species (ROS) and mitochondrial superoxide. A TUNUL assay kit was used to detect the level of apoptosis. Western blotting analysis was used to measure the expression of proteins. RESULTS: Treatment of isolated rat hearts with morphine prevented I/R-induced myocardial mitochondrial injury, which was inhibited by the selective EGFR inhibitor AG1478, suggesting that EGFR is involved in the mitochondrial protective effect of morphine under I/R conditions. In support of this hypothesis, the selective EGFR agonist epidermal growth factor (EGF) reduced mitochondrial morphological and functional damage similarly to morphine. Further study demonstrated that morphine may alleviate I/R-induced cardiac damage by inhibiting autophagy but not apoptosis. Morphine increased protein kinase B (Akt), extracellular regulated protein kinases (ERK) and signal transducer and activator of transcription-3 (STAT-3) phosphorylation, which was inhibited by AG1478, and EGF had similar effects, indicating that morphine may activate Akt, ERK, and STAT-3 via EGFR. Morphine and EGF increased intracellular reactive oxygen species (ROS) generation. This effect of morphine was inhibited by AG1478, indicating that morphine promotes intracellular ROS generation by activating EGFR. However, morphine did not increase ROS generation when cells were transfected with siRNA against EGFR. In addition, EGFR activity was markedly increased by morphine, but the effect of morphine was reversed by naltrindole. These results suggest that morphine may activate EGFR via δ-opioid receptor activation. CONCLUSIONS: Morphine may prevent I/R-induced myocardial mitochondrial damage by activating EGFR through δ-opioid receptors, in turn increasing RISK and SAFE pathway activity via intracellular ROS. Moreover, morphine may reduce myocardial injury by regulating autophagy but not apoptosis.

High-risk Surgically Resected Renal Cell Carcinoma: Is There a Role for Adjuvant VEGF-TKI Inhibitors?

The indications for adjuvant vascular endothelial growth factor-tyrosine kinase inhibitor (VEGF-TKI) agents after curative intent nephrectomy for renal cell carcinoma are still a matter of debate. The ASSURE, PROTECT and ATLAS trials have failed to meet their primary end-points. Conversely, S-TRAC has shown a disease free survival (DFS) benefit. To date, meta-analyses have repeatedly proved the absence of a clinical benefit, in term of DFS and overall survival (OS). Nevertheless, the results of the SORCE trial have been recently released and might add valuable information. We pooled the results of all five reported trials testing for any potential DFS and OS benefits associated with VEGF-TKI use. Interestingly, for pooled DFS we found a marginal positive hazard ratio (HR) of 0.92 (95% confidence interval [CI] 0.85-1.00; P-value = 0.049) in favor of adjuvant VEGF-TKI agents. This benefit was more pronounced for DFS in the sub-groups of only high-risk patients (HR: 0.89, 95% CI 0.80-0.99; P-value = 0.026), but less pronounced in clear-cell only subgroup (HR 0.92, 95% CI: 0.85-1.00; P-value = 0.044). Overall survival benefit was instead not reached. However, pooled relative risk for high-grade (grade ≥3 according to CTCAE classification) adverse events was irremediably high, 2.56 (95% CI: 2.15-3.04; P-value < 0.001). Given the marginal benefit in terms of DFS and the drawback of high-grade adverse events, even after the SORCE trial publication, adjuvant VEGF-TKIs therapy cannot be considered in the whole group of patients with non-metastatic high-risk renal cell carcinoma after surgery.

New Approaches for the Synthesis of Heterocyclic Compounds Derived from Cyclohexan-1,3-dione with Anti-proliferative Activities.

In the present work a series of heterocyclization reactions were adopted using cyclohexan-1,3-dione through its reaction with either furan-2-carbaldehyde or thiophene-2-carbaldehyde to give the corresponding ylidene derivatives 3a,b. The latter compounds underwent heterocyclization reactions to give thiophene and pyran derivatives 5a-d and 6a-d, respectively. Moreover, compounds 3a,b reacted with elemental sulfur and phenyl isothiocyanate to give the fused thiazole derivatives 8a,b. In addition, the reaction with either of hydrazine hydrate or phenylhydrazine has given the 4-hydrazono-4,5,6,7-tetrahydro-2H-indazole derivatives 10a-d, respectively. Similarly, the reaction of either 3a or 3b with hydroxylamine hydrochloride gave the 6,7-dihydrobenzo[c]isoxazol-4(5H)-one oxime derivatives 12a and 12b, respectively. Other fused heterocyclic compounds were produced and their structures were elucidated. Evaluation of the synthesized compounds against selected cancer cell lines was performed. The most active compounds were further evaluated against tyrosine kinases and Pim-1 kinase inhibitions.

Next-Generation Kinase Inhibitors Targeting Specific Biomarkers in Non-Small Cell Lung Cancer (NSCLC): A Recent Overview.

Lung cancer causes many deaths globally. Mutations in regulatory genes, irregularities in specific signal transduction events, or alterations of signalling pathways are observed in cases of non-small cell lung cancer (NSCLC). Over the past two decades, a few kinases have been identified, validated, and studied as biomarkers for NSCLC. Among them, EGFR, ALK, ROS1, MET, RET, NTRK, and BRAF are regarded as targetable biomarkers to cure and/or control the disease. In recent years, the US Food and Drug Administration (FDA) approved more than 15 kinase inhibitors targeting these NSCLC biomarkers. The kinase inhibitors significantly improved the progression-free survival (PFS) of NSCLC patients. Challenges still remain for metastatic diseases and advanced NSCLC cases. New discoveries of potent kinase inhibitors and rapid development of modern medical technologies will help to control NSCLC cases. This article provides an overview of the discoveries of various types of kinase inhibitors against NSCLC, along with medicinal chemistry aspects and related developments in next-generation kinase inhibitors that have been reported in recent years.

The Putative Glyoxalase 1 Inhibitor Piceatannol Exhibits Both Anxiolytic-like and Antitumor Effects in Mice.

BACKGROUND/AIM: This study aimed to determine the anxiolytic effect of a putative glyoxalase 1 inhibitor, piceatannol, as well as its antitumor activities on the stress-induced tumor growth of Lewis lung carcinoma. MATERIALS AND METHODS: The anxiolytic activities of piceatannol (1-30 mg/kg) were assessed using the elevated plus maze (EPM) test. We also evaluated the pharmacological modulation of stress-induced tumor growth; the mice were treated with piceatannol (3 and 30 mg/kg) from the 10th day till the 19th day after administration of the LLC cells. RESULTS: At the low dose (3 mg/kg), piceatannol significantly increased the time spent in the open arms of the EPM test when compared with the vehicle. At higher doses (30 mg/kg), it significantly suppressed the stress-induced enhancement of tumor growth. CONCLUSION: A low dose of piceatannol exerts an anxiolytic effect, and high doses have an antitumor effect.