Trametinib boosts palbociclib's efficacy in breast cancer via autophagy inhibition.

Breast cancer, a predominant global health issue, requires ongoing exploration of new therapeutic strategies. Palbociclib (PAL), a well-known cyclin-dependent kinase (CDK) inhibitor, plays a critical role in breast cancer treatment. While its efficacy is recognized, the interplay between PAL and cellular autophagy, particularly in the context of the RAF/MEK/ERK signaling pathway, remains insufficiently explored. This study investigates PAL's inhibitory effects on breast cancer using both in vitro (MCF7 and MDA-MB-468 cells) and in vivo (tumor-bearing nude mice) models. Aimed at elucidating the impact of PAL on autophagic processes and exploring the potential of combining it with trametinib (TRA), an MEK inhibitor, our research seeks to address the challenge of PAL-induced drug resistance. Our findings reveal that PAL significantly decreases the viability of MCF7 and MDA-MB-468 cells and reduces tumor size in mice while showing minimal cytotoxicity in MCF10A cells. However, PAL also induces protective autophagy, potentially leading to drug resistance via the RAF/MEK/ERK pathway activation. Introducing TRA effectively neutralized this autophagy, enhancing PAL's anti-tumor efficacy. A combination of PAL and TRA synergistically reduced cell viability and proliferation, and in vivo studies showed notable tumor size reduction. In conclusion, the PAL and TRA combination emerges as a promising strategy for overcoming PAL-induced resistance, offering a new horizon in breast cancer treatment.

Regorafenib synergizes with TAS102 against multiple gastrointestinal cancers and overcomes cancer stemness, trifluridine-induced angiogenesis, ERK1/2 and STAT3 signaling regardless of KRAS or BRAF mutational status.

Single-agent TAS102 (trifluridine/tipiracil) and regorafenib are FDA-approved treatments for metastatic colorectal cancer (mCRC). We previously reported that regorafenib combined with a fluoropyrimidine can delay disease progression in clinical case reports of multidrug-resistant mCRC patients. We hypothesized that the combination of TAS102 and regorafenib may be active in CRC and other gastrointestinal (GI) cancers and may in the future provide a treatment option for patients with advanced GI cancer. We investigated the therapeutic effect of TAS102 in combination with regorafenib in preclinical studies employing cell culture, colonosphere assays that enrich for cancer stem cells, and in vivo. TAS102 in combination with regorafenib has synergistic activity against multiple GI cancers in vitro including colorectal and gastric cancer, but not liver cancer cells. TAS102 inhibits colonosphere formation and this effect is potentiated by regorafenib. In vivo anti-tumor effects of TAS102 plus regorafenib appear to be due to anti-proliferative effects, necrosis and angiogenesis inhibition. Growth inhibition by TAS102 plus regorafenib occurs in xenografted tumors regardless of p53, KRAS or BRAF mutations, although more potent tumor suppression was observed with wild-type p53. Regorafenib significantly inhibits TAS102-induced angiogenesis and microvessel density in xenografted tumors, as well inhibits TAS102-induced ERK1/2 activation regardless of RAS or BRAF status in vivo. TAS102 plus regorafenib is a synergistic drug combination in preclinical models of GI cancer, with regorafenib suppressing TAS102-induced increase in microvessel density and p-ERK as contributing mechanisms. The TAS102 plus regorafenib drug combination may be further tested in gastric and other GI cancers.

Quercetin inhibits the epithelial-mesenchymal transition and reverses CDK4/6 inhibitor resistance in breast cancer by regulating circHIAT1/miR-19a-3p/CADM2 axis.

Breast cancer (BC) cells have a high risk of metastasis due to epithelial-mesenchymal transition (EMT). Palbociclib (CDK4/6 inhibitor) is an approved drug for BC treatment. However, the drug resistance and metastasis can impair the treatment outcome of Palbociclib. Understanding the mechanisms of EMT and Palbociclib drug resistance in BC is conducive to the formulation of novel therapeutic strategy. Here, we investigated the role of circHIAT1/miR-19a-3p/CADM2 axis in modulating EMT and Palbociclib resistance in BC. circHIAT1 and CADM2 were down-regulated in BC tissues and cell lines, and miR-19a-3p showed an up-regulation. circHIAT1 could interact with miR-19a-3p and suppress its activity, while miR-19a-3p functioned to negatively regulate CADM2. Forced over-expression of circHIAT1 could impaired the EMT status and migratory ability of BC cells, and this effect was inhibited by miR-19a-3p mimic. In addition, we also generated Palbociclib resistant BC cells, and showed that circHIAT1 and CADM2 were down-regulated in the resistant BC cells while miR-19a-3p showed an up-regulation. Forced circHIAT1 over-expression re-sensitized BC cells to Palbociclib treatment. Quercetin, a bioactive flavonoid, could suppressed the migration and invasion of BC cells, and re-sensitized BC cells to Palbociclib. The anti-cancer effect of quercetin could be attributed to its regulatory effect on circHIAT1/miR-19a-3p/CADM2 axis. In vivo tumorigenesis experiment further revealed that quercetin administration enhanced the anti-cancer effect of Palbociclib, an effect was dependent on the up-regulation of circHIAT1 by quercetin. In summary, this study identified quercetin as a potential anti-cancer compound to reverse Palbociclib resistance and impair EMT in BC cells by targeting circHIAT1/miR-19a-3p/CADM2 axis.

A Kinome-Wide Synthetic Lethal CRISPR/Cas9 Screen Reveals That mTOR Inhibition Prevents Adaptive Resistance to CDK4/CDK6 Blockade in HNSCC.

The comprehensive genomic analysis of the head and neck squamous cell carcinoma (HNSCC) oncogenome revealed the frequent loss of p16INK4A (CDKN2A) and amplification of cyclin D1 genes in most human papillomavirus-negative HNSCC lesions. However, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have shown modest effects in the clinic. The aberrant activation of the PI3K/mTOR pathway is highly prevalent in HNSCC, and recent clinical trials have shown promising clinical efficacy of mTOR inhibitors (mTORi) in the neoadjuvant and adjuvant settings but not in patients with advanced HNSCC. By implementing a kinome-wide CRISPR/Cas9 screen, we identified cell-cycle inhibition as a synthetic lethal target for mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergism in HNSCC-derived cells in vitro and in vivo. Remarkably, we found that an adaptive increase in cyclin E1 (CCNE1) expression upon palbociclib treatment underlies the rapid acquired resistance to this CDK4/6 inhibitor. Mechanistically, mTORi inhibits the formation of eIF4G-CCNE1 mRNA complexes, with the consequent reduction in mRNA translation and CCNE1 protein expression. Our findings suggest that mTORi reverts the adaptive resistance to palbociclib. This provides a multimodal therapeutic option for HNSCC by cotargeting mTOR and CDK4/6, which in turn may halt the emergence of palbociclib resistance. SIGNIFICANCE: A kinome-wide CRISPR/Cas9 screen identified cell-cycle inhibition as a synthetic lethal target of mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergistic effects in HNSCC. Mechanistically, mTORis inhibited palbociclib-induced increase in CCNE1.

The Structure-Antiproliferative Activity Relationship of Pyridine Derivatives.

Pyridine, a compound with a heterocyclic structure, is a key player in medicinal chemistry and drug design. It is widely used as a framework for the design of biologically active molecules and is the second most common heterocycle in FDA-approved drugs. Pyridine is known for its diverse biological activity, including antituberculosis, antitumor, anticoagulant, antiviral, antimalarial, antileishmania, anti-inflammatory, anti-Alzheimer's, antitrypanosomal, antimalarial, vasodilatory, antioxidant, antimicrobial, and antiproliferative effects. This review, spanning from 2022 to 2012, involved the meticulous identification of pyridine derivatives with antiproliferative activity, as indicated by their minimum inhibitory concentration values (IC50) against various cancerous cell lines. The aim was to determine the most favorable structural characteristics for their antiproliferative activity. Using computer programs, we constructed and calculated the molecular descriptors and analyzed the electrostatic potential maps of the selected pyridine derivatives. The study found that the presence and positions of the -OMe, -OH, -C=O, and NH2 groups in the pyridine derivatives enhanced their antiproliferative activity over the cancerous cellular lines studied. Conversely, pyridine derivatives with halogen atoms or bulky groups in their structures exhibited lower antiproliferative activity.

Combination of dual JAK/HDAC inhibitor with regorafenib synergistically reduces tumor growth, metastasis, and regorafenib-induced toxicity in colorectal cancer.

BACKGROUND: Treatment with regorafenib, a multiple-kinase inhibitor, to manage metastatic colorectal cancers (mCRCs) shows a modest improvement in overall survival but is associated with severe toxicities. Thus, to reduce regorafenib-induced toxicity, we used regorafenib at low concentration along with a dual JAK/HDAC small-molecule inhibitor (JAK/HDACi) to leverage the advantages of both JAK and HDAC inhibition to enhance antitumor activity. The therapeutic efficacy and safety of the combination treatment was evaluated with CRC models. METHODS: The cytotoxicity of JAK/HDACi, regorafenib, and their combination were tested with normal colonic and CRC cells exhibiting various genetic backgrounds. Kinomic, ATAC-seq, RNA-seq, cell cycle, and apoptosis analyses were performed to evaluate the cellular functions/molecular alterations affected by the combination. Efficacy of the combination was assessed using patient-derived xenograft (PDX) and experimental metastasis models of CRC. To evaluate the interplay between tumor, its microenvironment, and modulation of immune response, MC38 syngeneic mice were utilized. RESULTS: The combination therapy decreased cell viability; phosphorylation of JAKs, STAT3, EGFR, and other key kinases; and inhibited deacetylation of histone H3K9, H4K8, and alpha tubulin proteins. It induced cell cycle arrest at G0-G1 phase and apoptosis of CRC cells. Whole transcriptomic analysis showed that combination treatment modulated molecules involved in apoptosis, extracellular matrix-receptor interaction, and focal adhesion pathways. It synergistically reduces PDX tumor growth and experimental metastasis, and, in a syngeneic mouse model, the treatment enhances the antitumor immune response as evidenced by higher infiltration of CD45 and cytotoxic cells. Pharmacokinetic studies showed that combination increased the bioavailability of regorafenib. CONCLUSIONS: The combination treatment was more effective than with regorafenib or JAK/HDACi alone, and had minimal toxicity. A clinical trial to evaluate this combination for treatment of mCRCs is warranted.

The Chromenopyridine Scaffold: A Privileged Platform in Drug Design.

The chromenopyridine scaffold represents an important class of heterocyclic compounds exhibiting a broad spectrum of biological properties. This review describes novel and efficient procedures for the synthesis of this scaffold. Herein, several methods were detailed and grouped according to their starting material (e.g., salicylaldehydes, chromones, chromanones and coumarins) and respective biological activity, when reported. This review highlights the potential of the reported synthetic strategies for preparing chromenopyridine derivatives with promising biological activity, paving the way for further developments in drug discovery.

Identification of Novel GANT61 Analogs with Activity in Hedgehog Functional Assays and GLI1-Dependent Cancer Cells.

Aberrant activation of hedgehog (Hh) signaling has been implicated in various cancers. Current FDA-approved inhibitors target the seven-transmembrane receptor Smoothened, but resistance to these drugs has been observed. It has been proposed that a more promising strategy to target this pathway is at the GLI1 transcription factor level. GANT61 was the first small molecule identified to directly suppress GLI-mediated activity; however, its development as a potential anti-cancer agent has been hindered by its modest activity and aqueous chemical instability. Our study aimed to identify novel GLI1 inhibitors. JChem searches identified fifty-two compounds similar to GANT61 and its active metabolite, GANT61-D. We combined high-throughput cell-based assays and molecular docking to evaluate these analogs. Five of the fifty-two GANT61 analogs inhibited activity in Hh-responsive C3H10T1/2 and Gli-reporter NIH3T3 cellular assays without cytotoxicity. Two of the GANT61 analogs, BAS 07019774 and Z27610715, reduced Gli1 mRNA expression in C3H10T1/2 cells. Treatment with BAS 07019774 significantly reduced cell viability in Hh-dependent glioblastoma and lung cancer cell lines. Molecular docking indicated that BAS 07019774 is predicted to bind to the ZF4 region of GLI1, potentially interfering with its ability to bind DNA. Our findings show promise in developing more effective and potent GLI inhibitors.

Ruthenium p-Cymene Complexes Incorporating Substituted Pyridine-Quinoline-Based Ligands: Synthesis, Characterization, and Cytotoxic Properties.

Organometallic complexes of the formula [Ru(N^N)(p-cymene)Cl][X] (N^N = bidentate polypyridyl ligands, p-cymene = 1-methyl-4-(1-methylethyl)-benzene, X = counter anion), are currently studied as possible candidates for the potential treatment of cancer. Searching for new organometallic compounds with good to moderate cytotoxic activities, a series of mononuclear water-soluble ruthenium(II)-arene complexes incorporating substituted pyridine-quinoline ligands, with pending -CH2OH, -CO2H and -CO2Me groups in the 4-position of quinoline ring, were synthesized, for the first time, to study their possible effect to modulate the activity of the ruthenium p-cymene complexes. These include the [Ru(η6-p-cymene)(pqhyme)Cl][X] (X = Cl- (1-Cl), PF6- (1-PF6), pqhyme = 4-hydroxymethyl-2-(pyridin-2-yl)quinoline), [Ru(η6-p-cymene)(pqca)Cl][Cl] ((2-Cl), pqca = 4-carboxy-2-(pyridin-2-yl)quinoline), and [Ru(η6-p-cymene)(pqcame)Cl][X] (X = Cl- (3-Cl), PF6- (3-PF6), pqcame = 4-carboxymethyl-2-(pyridin-2-yl)quinoline) complexes, respectively. Identification of the complexes was based on multinuclear NMR and ATR-IR spectroscopic methods, elemental analysis, conductivity measurements, UV-Vis spectroscopic, and ESI-HRMS techniques. The solid-state structures of 1-PF6 and 3-PF6 have been elucidated by single-crystal X-ray diffraction revealing a three-legged piano stool geometry. This is the first time that the in vitro cytotoxic activities of these complexes are studied. These were conducted in HEK293T (human embryonic kidney cells) and HeLa cells (cervical cancer cells) via the MTT assay. The results show poor in vitro anticancer activities for the HeLa cancer cell lines and 3-Cl proved to be the most potent (IC50 > 80 µΜ). In both cell lines, the cytotoxicity of the ligand precursor pqhyme is significantly higher than that of cisplatin.

The Study of the Effectiveness of Ethylmethylhydroxypyridine Succinate in Acute Alcohol Intoxication.

The effectiveness of ethylmethylhydroxypyridine succinate (EMHPS) in acute alcohol intoxication was tested in a study on SPF male outbred ICR mice. Ethanol (concentration 40%) was administered to animals once intraperitoneally at a dose of 4 g/kg. Control animals were injected with saline in an equivalent volume. In 15 min after the administration of alcohol, the animals were injected intravenously or intramuscularly with EMHPS at a dose of 50 or 100 mg/kg or with saline via the same route in an equivalent volume. Animal behavior was tested 3 and 24 h later after administration of the substances. After 3 and 24 h, mice in the pathological control groups developed semiptosis, the gait and the turning over reflex were impaired, the strength of the hind limbs decreased and the distance between the hind limbs increased when landing; in the open-field test, the latency of the first movement increased, and the number of rearing postures decreased. Intravenous and intramuscular administration of EMHPS in doses of 50 and 100 mg/kg had a pronounced antitoxic and neuroprotective effect in acute alcohol intoxication: all studied parameters did not differ significantly from the control.

Inhibition of M. tuberculosis and human ATP synthase by BDQ and TBAJ-587.

Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase1,2. However, BDQ also inhibits human ATP synthase3. At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers' understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs.

Comparative biological activity of palbociclib and ribociclib in hormone receptor-positive breast cancer.

This study examines the biological effects of palbociclib and ribociclib in hormone receptor-positive breast cancer, pivotal to the HARMONIA prospective phase III clinical trial. We explore the downstream impacts of these CDK4/6 inhibitors, focusing on cell lines and patient-derived tumor samples. We treated HR+ breast cancer cell lines (T47D, MCF7, and BT474) with palbociclib or ribociclib (100 nM or 500 nM), alone or combined with fulvestrant (1 nM), over periods of 24, 72, or 144 h. Our assessments included PAM50 gene expression, RB1 phosphorylation, Lamin-B1 protein levels, and senescence-associated ß-galactosidase activity. We further analyzed PAM50 gene signatures from the CORALLEEN and NeoPalAna phase II trials. Both CDK4/6 inhibitors similarly inhibited proliferation across the cell lines. At 100 nM, both drugs partially reduced p-RB1, with further decreases at 500 nM over 144 h. Treatment led to reduced Lamin-B1 expression and increased senescence-associated ß-galactosidase activity. Both drugs enhanced Luminal A and reduced Luminal B and proliferation signatures at both doses. However, the HER2-enriched signature significantly diminished only at the higher dose of 500 nM. Corresponding changes were observed in tumor samples from the CORALLEEN and NeoPalAna studies. At 2 weeks of treatment, both drugs significantly reduced the HER2-enriched signature, but at surgery, this reduction was consistent only with ribociclib. Our findings suggest that while both CDK4/6 inhibitors effectively modulate key biological pathways in HR+/HER2- breast cancer, nuances in their impact, particularly on the HER2-enriched signature, are dose-dependent, influenced by the addition of fulvestrant and warrant further investigation.

O-GlcNAcylation of MITF regulates its activity and CDK4/6 inhibitor resistance in breast cancer.

Cyclin-dependent kinases 4 and 6 (CDK4/6) play a pivotal role in cell cycle and cancer development. Targeting CDK4/6 has demonstrated promising effects against breast cancer. However, resistance to CDK4/6 inhibitors (CDK4/6i), such as palbociclib, remains a substantial challenge in clinical settings. Using high-throughput combinatorial drug screening and genomic sequencing, we find that the microphthalmia-associated transcription factor (MITF) is activated via O-GlcNAcylation by O-GlcNAc transferase (OGT) in palbociclib-resistant breast cancer cells and tumors. Mechanistically, O-GlcNAcylation of MITF at Serine 49 enhances its interaction with importin α/ß, thus promoting its translocation to nuclei, where it suppresses palbociclib-induced senescence. Inhibition of MITF or its O-GlcNAcylation re-sensitizes resistant cells to palbociclib. Moreover, clinical studies confirm the activation of MITF in tumors from patients who are palbociclib-resistant or undergoing palbociclib treatment. Collectively, our studies shed light on the mechanism regulating palbociclib resistance and present clinical evidence for developing therapeutic approaches to treat CDK4/6i-resistant breast cancer patients.

Discovering novel derivatives of STAT3 and HDAC inhibitors with anti-tumor activity.

In modern cancer therapy, blockage of more than one target is a standard approach, and there are already many dual-target drugs that can achieve multiple inhibition through a single molecule. Herein, we designed and synthesized a series of novel derivatives with signal transducer and activator of transcription 3 (STAT3) and histone deacetylase (HDAC) inhibitory activity through strategy of combining pharmacophore based on the STAT3 inhibitor E28 and HDAC inhibitor MS-275. Among them, compound 24 (IC50 = 8.22 ± 0.27 µM) showed better anti-tumor activity than the clinical Class I HDAC inhibitor MS-275 (IC50 = 14.65 ± 0.24 µM) in MCF-7 breast cancer cells. Furthermore, the dual inhibition to HDAC and STAT3 of compound 24 was validated by western blot analysis. The study provides new tool compounds for further exploration of STAT3-HDAC pathway inhibitor achieved with a single molecule.

Capturing the Effects of Single Atom Substitutions on the Inhibition Efficiency of Glycogen Synthase Kinase-3ß Inhibitors via Markov State Modeling and Experiments.

Small modifications in the chemical structure of ligands are known to dramatically change their ability to inhibit the activity of a protein. Unraveling the mechanisms that govern these dramatic changes requires scrutinizing the dynamics of protein-ligand binding and unbinding at the atomic level. As an exemplary case, we have studied Glycogen Synthase Kinase-3ß (GSK-3ß), a multifunctional kinase that has been implicated in a host of pathological processes. As such, there is a keen interest in identifying ligands that inhibit GSK-3ß activity. One family of compounds that are highly selective and potent inhibitors of GSK-3ß is exemplified by a molecule termed COB-187. COB-187 consists of a five-member heterocyclic ring with a thione at C2, a pyridine substituted methyl at N3, and a hydroxyl and phenyl at C4. We have studied the inhibition of GSK-3ß by COB-187-related ligands that differ in a single heavy atom from each other (either in the location of nitrogen in their pyridine ring, or with the pyridine ring replaced by a phenyl ring), or in the length of the alkyl group joining the pyridine and the N3. The inhibition experiments show a large range of half-maximal inhibitory concentration (IC50) values from 10 nM to 10 µM, implying that these ligands exhibit vastly different propensities to inhibit GSK-3ß. To explain these differences, we perform Markov State Modeling (MSM) using fully atomistic simulations. Our MSM results are in excellent agreement with the experiments in that they accurately capture differences in the binding propensities of the ligands. The simulations show that the binding propensities are related to the ligands' ability to attain a compact conformation where their two aromatic rings are spatially close. We rationalize this result by sampling numerous binding and unbinding events via funnel metadynamics simulations, which show that indeed while approaching the bound state, the ligands prefer to be in their compact conformation. We find that the presence of nitrogen in the aromatic ring increases the probability of attaining the compact conformation. Protein-ligand binding is understood to be dictated by the energetics of interactions and entropic factors, like the release of bound water from the binding pockets. This work shows that changes in the conformational distribution of ligands due to atom-level modifications in the structure play an important role in protein-ligand binding.

Targeting specific DNA G-quadruplexes with CRISPR-guided G-quadruplex-binding proteins and ligands.

Despite the demonstrated importance of DNA G-quadruplexes (G4s) in health and disease, technologies to readily manipulate specific G4 folding for functional analysis and therapeutic purposes are lacking. Here we employ G4-stabilizing protein/ligand in conjunction with CRISPR to selectively facilitate single or multiple targeted G4 folding within specific genomic loci. We demonstrate that fusion of nucleolin with a catalytically inactive Cas9 can specifically stabilize G4s in the promoter of oncogene MYC and muscle-associated gene Itga7 as well as telomere G4s, leading to cell proliferation arrest, inhibition of myoblast differentiation and cell senescence, respectively. Furthermore, CRISPR can confer intra-G4 selectivity to G4-binding compounds pyridodicarboxamide and pyridostatin. Compared with traditional G4 ligands, CRISPR-guided biotin-conjugated pyridodicarboxamide enables a more precise investigation into the biological functionality of de novo G4s. Our study provides insights that will enhance understanding of G4 functions and therapeutic interventions.

Inhibition of the TCF12/VSIG4 axis by palbociclib diminishes the proliferation and migration of glioma cells and decreases the M2 polarization of glioma-associated microglia.

The CDK4/CDK6 inhibitor palbociclib has shown the encouraging promise in the treatment of glioma. Here, we elucidated how palbociclib exerts suppressive functions in the M2 polarization of glioma-related microglia and the progression of glioma. Xenograft experiments were used to evaluate the function in vivo. The mRNA levels of transcription factor 12 (TCF12) and VSIG4 were detected by RT-qPCR, and their protein levels were assessed by immunoblotting. Cell migration was tested by wound-healing assay. Cell cycle distribution and M1/M2 microglia phenotype analysis were performed by flow cytometry. The levels of IFN-γ, TNF-α, IL-6，and TGF-ß were measured by ELISA. The TCF12/VSIG4 association was verified by luciferase reporter and chromatin immunoprecipitation (ChIP) assays. In U251 and LN229 glioma cells, TCF12 and VSIG4 were overexpressed, and palbociclib reduced their expression levels. TCF12 upregulation enhanced the proliferation and migration of glioma cells and the M2 polarization of glioma-associated microglia in vitro as well as the tumorigenicity of U251 glioma cells in vivo, which could be reversed by palbociclib. Mechanistically, TCF12 could enhance VSIG4 transcription and expression by binding to the VSIG4 promoter. TCF12 deficiency led to repression in glioma cell proliferation and migration as well as microglia M2 polarization, which could be abolished by increased VSIG4 expression. Our study reveals the novel TCF12/VSIG4 axis responsible for the efficacy of palbociclib in combating glioma, offering a rationale for the application of palbociclib in glioma treatment.

Unravelling nicotinic receptor and ligand features underlying neonicotinoid knockdown actions on the malaria vector mosquito Anopheles gambiae.

With the spread of resistance to long-established insecticides targeting Anopheles malaria vectors, understanding the actions of compounds newly identified for vector control is essential. With new commercial vector-control products containing neonicotinoids under development, we investigate the actions of 6 neonicotinoids (imidacloprid, thiacloprid, clothianidin, dinotefuran, nitenpyram and acetamiprid) on 13 Anopheles gambiae nicotinic acetylcholine receptor (nAChR) subtypes produced by expression of combinations of the Agα1, Agα2, Agα3, Agα8 and Agß1 subunits in Xenopus laevis oocytes, the Drosophila melanogaster orthologues of which we have previously shown to be important in neonicotinoid actions. The presence of the Agα2 subunit reduces neonicotinoid affinity for the mosquito nAChRs, whereas the Agα3 subunit increases it. Crystal structures of the acetylcholine binding protein (AChBP), an established surrogate for the ligand-binding domain, with dinotefuran bound, shows a unique target site interaction through hydrogen bond formation and CH-N interaction at the tetrahydrofuran ring. This is of interest as dinotefuran is also under trial as the toxic element in baited traps. Multiple regression analyses show a correlation between the efficacy of neonicotinoids for the Agα1/Agα2/Agα8/Agß1 nAChR, their hydrophobicity and their rate of knockdown of adult female An. gambiae, providing new insights into neonicotinoid features important for malaria vector control.

Deciphering the Interplay: Thieno[2,3-b]pyridine's Impact on Glycosphingolipid Expression, Cytotoxicity, Apoptosis, and Metabolomics in Ovarian Tumor Cell Lines.

Ovarian cancer is among the most prevalent causes of mortality among women. Despite improvements in diagnostic methods, non-specific symptoms and delayed gynecological exams can lead to late-stage ovarian tumor discovery. In this study, the effect of an anti-cancer compound, 3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide (Compound 1), was examined. The impacts of cytotoxicity, apoptosis, and metabolomic changes in ovarian cancer cell lines SK-OV-3 and OVCAR-3, as well as glycosphingolipid (GSL) expression, on cancer stem cells (CSCs), marked as CD49f+, and non-CSCs (CD49f-) were explored. Treatment with Compound 1 reduced the percentage of CSCs compared to non-treated cells (p < 0.001). The functional impact of eight GSLs on CSCs and non-CSCs was examined using flow cytometry. The glycophenotype changed in both cell lines, with increases or decreases in its expression, after the treatment. These findings raise the possibility of specifically targeting CSCs in ovarian cancer therapy. Additionally, treatment with Compound 1 resulted in statistically meaningful increased apoptosis, including both early and late apoptosis (p < 0.001), suggesting a pivotal role in initiating programmed cell death by the apoptotic pathway. The analysis revealed that the metabolic activity of treated cancer cells was lower compared to those of the control group (p < 0.001).

APC/C prevents a noncanonical order of cyclin/CDK activity to maintain CDK4/6 inhibitor-induced arrest.

Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C), although the APC/C substrates whose degradation restrains G1-S progression are not fully known. The APC/C is also active in arrested cells that exited the cell cycle, but it is not clear whether APC/C maintains all types of arrest. Here, by expressing the APC/C inhibitor, EMI1, we show that APC/C activity is essential to prevent S phase entry in cells arrested by pharmacological cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition (Palbociclib). Thus, active protein degradation is required for arrest alongside repressed cell cycle gene expression. The mechanism of rapid and robust arrest bypass from inhibiting APC/C involves CDKs acting in an atypical order to inactivate retinoblastoma-mediated E2F repression. Inactivating APC/C first causes mitotic cyclin B accumulation which then promotes cyclin A expression. We propose that cyclin A is the key substrate for maintaining arrest because APC/C-resistant cyclin A, but not cyclin B, is sufficient to induce S phase entry. Cells bypassing arrest from CDK4/6 inhibition initiate DNA replication with severely reduced origin licensing. The simultaneous accumulation of S phase licensing inhibitors, such as cyclin A and geminin, with G1 licensing activators disrupts the normal order of G1-S progression. As a result, DNA synthesis and cell proliferation are profoundly impaired. Our findings predict that cancers with elevated EMI1 expression will tend to escape CDK4/6 inhibition into a premature, underlicensed S phase and suffer enhanced genome instability.