Journal of Pharmacology and Experimental Therapeutics最新文献

Androgen deprivation is the standard treatment for patients with prostate cancer. However, the disease eventually progresses as castration-resistant prostate cancer (CRPC). Enzalutamide, an androgen receptor inhibitor, is a typical drug for treating CRPC and with continuous reliance on the drug, can lead to enzalutamide resistance. This highlights the necessity for developing novel therapeutic targets to combat the gain of resistance. Metformin has been recently investigated for its potential antitumorigenic effects in many cancer types. In this study, we used enzalutamide and metformin in combination to explore the possible rescued efficacy of enzalutamide in the treatment of enzalutamide-resistant CRPC. We first tested the effects of this combination treatment on cell viability, drug synergy, and cell proliferation in enzalutamide-resistant CRPC cell lines. After combination treatment, we observed a decrease in cell proliferation and viability as well as a synergistic effect of both enzalutamide and metformin in vitro. Following these results, we sought to explore how combination treatment affected mitochondrial fitness using mitochondrial stress test analysis and mitochondrial membrane potential shifts due to metformin's action in inhibiting complex I of oxidative phosphorylation. We employed 2 different strategies for in vivo testing using 22Rv1 and LuCaP35CR xenograft models. Finally, RNA sequencing revealed a potential link in the downregulation of rat sarcoma-mitogen-activated protein kinase signaling following combination treatment. SIGNIFICANCE STATEMENT: Increasing evidence suggests that oxidative phosphorylation might play a critical role in the development of resistance to cancer therapy. This study showed that targeting oxidative phosphorylation with metformin can enhance the efficacy of enzalutamide in castration-resistant prostate cancer in vitro.

Through its pathological and genetic association with Parkinson disease (PD), α-synuclein (α-syn) remains a favorable therapeutic target that is being investigated using various modalities, including many passive immunotherapy approaches clinically targeting different forms of α-syn and epitopes. Although published studies from some immunotherapy trials have demonstrated engagement in plasma, none has shown direct drug-antigen interactions in the disease-relevant compartment, the central nervous system. Cinpanemab (BIIB054) selectively targets pathological aggregated α-syn with low-affinity binding to monomeric forms. The avidity-driven binding, low drug concentration, and the very low α-syn levels, plus its heterogeneous nature in cerebrospinal fluid (CSF), made it impossible to measure drug-target interactions by conventional assays. Here we overcame these challenges by using zero-length crosslinking to stabilize the BIIB054-α-syn complexes and then quantified the crosslinked complexes using a Meso Scale Discovery electrochemiluminescence assay. CSF samples from healthy volunteers (HVs, n = 46) and individuals with PD (PD, n = 18) from study 228HV101 (phase 1 clinical trial of BIIB054) demonstrated dose- and time-dependent binding of cinpanemab to α-syn with measurable complexes detected at doses ≥15 mg/kg. Complex formation displayed a direct positive correlation to drug concentration (Spearman rank correlation = 0.8295 [HV], 0.8032 [PD] P < .0001 [HV, PD]). The observed binding of cinpanemab to α-syn in CSF is consistent with its low intrinsic affinity for α-syn monomer and provides evidence that the drug is behaving with expected binding dynamics in the central nervous system compartment. SIGNIFICANCE STATEMENT: A zero-length crosslinking method with Meso Scale Discovery detection was developed to enable quantification of cinpanemab-α-synuclein (α-syn) complexes in clinical cerebrospinal fluid samples by preventing signal loss caused by their rapid dissociation. Observed dose- and time-dependent binding was consistent with cinpanemab's affinity for α-syn and provided confidence the drug had engaged its target at the desired site of action. This is the first demonstration of α-syn binding by an antibody in clinical samples from the central nervous system.

Cardiovascular disease (CVD) remains one of the leading causes of death worldwide. Aberrant platelet function mediates fibrin(ogen)-rich thrombi that lead to occlusive thrombi associated with mortality. The receptor, TREM-like transcript-1 (TLT-1), stored in the platelet α-granules and released upon platelet activation, binds fibrinogen and von Willebrand factor. Once it is released from platelets, TLT-1 is a potential therapeutic target to prevent the thrombosis associated with CVD. Here we designed an assay to screen a compound library of small molecules inhibitors. Human embryonic kidney (HEK)-293 cells stably transfected with a full-length human treml-1 construct were used to screen library of 800 compounds, for inhibition of TLT-1 to fibrinogen binding in an attachment assay using crystal violet staining. The possible cytotoxicity of the best compounds was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide MTT and calcein AM staining assays. We demonstrated that the addition of TLT-1 to HEK-293 cells increases cell adhesion by more than 2-fold. We identified ∼80 compounds that inhibit binding by more than 80%. We further tested the top compounds and confirmed that reduction of hTLT-1 to fibrinogen bound in the top compounds was not caused by cytotoxicity, as per colorimetric and fluorescent viability assays. Four compounds were identified as potential small molecule inhibitors, one of which, BM-8372, demonstrated significant effect in platelet aggregation and spreading assays. SIGNIFICANCE STATEMENT: Triggering receptor expressed in myeloid cells-like transcript-1 (TLT-1) is a key platelet receptor that binds fibrinogen and mediates clot formation. The developed assay successfully screened 800 small molecules, pinpointing ∼80 potent inhibitors that reduce TLT-1 binding by over 80%. Importantly, the study rigorously rules out cytotoxicity concerns, affirming the therapeutic potential of the identified compounds. By elucidating TLT-1's role and presenting promising inhibitors, this research offers a significant stride toward developing novel strategies to combat cardiovascular disease-related thrombosis.

Cancer patients have an increased risk of venous thromboembolism, which is their second cause of death after disease progression itself. Several thrombotic risk factors coexist in cancer patients, including the ability of both cancer and tumoral microenvironment's cells to directly or indirectly activate platelets and the enzymes of the coagulation cascade, resulting in a hypercoagulable state of blood. This narrative review gives an overview of the main mechanisms leading to venous thromboembolism in cancer patients, including the role that platelets and the clotting proteins may have in tumor growth and metastasis. Of note, the hemostatic balance is altered in cancer patients who may, next to a thrombosis tendency, also have an increased risk of bleeding. To highlight the complexity and the precariousness of the hemostatic balance of these patients, we discuss 2 specific gastrointestinal malignancies: hepatocellular carcinoma, which is frequently associated with liver cirrhosis, a condition that causes profound alterations of hemostasis, and colorectal cancer, which is characterized by a fragile mucosa that is prone to bleeding. Understanding the molecular mechanisms of cancer-associated thrombosis may give a unique opportunity to develop new innovative drugs, acting differently on distinct pathways and potentially allowing to reduce the risk of bleeding related to antithrombotic therapies. SIGNIFICANCE STATEMENT: The topic is significant because understanding the molecular mechanisms leading to cancer-associated thrombosis and bleeding, focusing on gastrointestinal malignancies, enables the development of more rationale and innovative antithrombotic strategies for cancer-associated thrombosis. Eventually, this will support an improved and patient-tailored antithrombotic management in vulnerable oncologic patients.

Triple-negative breast cancer (TNBC) is characterized by high mortality rates, primarily due to its propensity for metastasis. Addressing this challenge necessitates the development of effective antimetastatic therapies. This study aimed to identify natural compounds with potential antimetastatic properties mainly based on the high-throughput phenotypic screening system. This system, utilizing luciferase reporter gene assays combined with scratch wound assays, evaluates compounds based on their influence on the epithelial-mesenchymal transition (EMT) marker E-cadherin. Through this approach, aurovertin B (AVB) was revealed to have significant antimetastatic capability. Notably, AVB exhibited substantial metastasis suppression in many TNBC cell lines, including MDA-MB-231, HCC1937, and 4T1. Also, its remarkable antimetastatic activity was demonstrated in vivo via the orthotopic breast cancer mouse model. Further exploration revealed a pronounced association between AVB-induced upregulation of dual-specificity phosphatase 1 (DUSP1) and its inhibitory effect on TNBC metastasis. Additionally, microarray analysis conducted to elucidate the underlying mechanism of the AVB-DUSP1 interaction identified activating transcription factor 3 (ATF3) as a critical transcription factor instrumental in DUSP1 transcriptional activation. This discovery, coupled with observations of enhanced ATF3-DUSP1 expression and consequent reduction in TNBC metastatic foci in response to AVB, provides novel insights into the metastatic mechanisms of TNBC. SIGNIFICANCE STATEMENT: This study constructs a high-throughput phenotypic screening system utilizing epithelial-mesenchymal transition marker E-cadherin promoter luciferase reporter gene combined with scratch wound assays. Aurovertin B was revealed to possess significant antimetastatic activity through this approach, which was further demonstrated via in vivo and in vitro experiments. The discovery of the regulatory role of the ATF3-DUSP1 pathway enriches our understanding of TNBC metastasis mechanism and suggests the potential of ATF3 and DUSP1 as biomarkers for diagnosing TNBC metastasis.

We examined the effects of stress on the indomethacin (IND)-induced gastric antral ulcer formation in refed mice. Male mice underwent refeeding of diet for 2 hours after a fast for 22 hours, followed by IND injection; the lesion index was measured 24 hours later. Mice also underwent a defined diet for 2 hours following a fast for 22 hours, and the stomachs were collected 1.5 hours later. We then measured the volume and the bile acid concentrations of the gastric contents. Mice underwent restraint stress (RS) in a cylindrical plastic tube for 60 minutes, or treatment with corticotropin-releasing factor (CRF), following refeeding of diet for 2 hours. We then examined the effects of RS and CRF on the lesion index, gastric emptying, and duodenogastric bile reflux. The effects of receptor antagonists for CRF₂ (astressin-2B), CRF₁, 5-hydroxytriptamine 3 (ondansetron), dopamine 2 (haloperidol), and cholecystokinin 1 (lorglumide) on the effects of RS or CRF were examined. IND (10 mg/kg, s.c.) induced pronounced lesions in the antrum. RS and CRF (30 μg/kg, i.p.) increased the severity of the antral lesions accompanied by an increase in gastric volume and concentration of bile acids. These effects of RS and peripheral CRF were significantly inhibited by pretreatment with astressin-2B, ondansetron, haloperidol, and lorglumide, but not by the CRF₁ receptor antagonist. This study suggests that RS increases the severity of IND-induced gastric antral ulcers associated with gastroparesis and enhanced bile reflux via activation of peripheral CRF₂, 5-hydroxytriptamine 3, and cholecystokinin 1 receptors with central dopamine 2 receptor, but not by CRF₁ receptor. SIGNIFICANCE STATEMENT: Restraint stress worsens nonsteroidal anti-inflammatory drugs-induced antral ulcers due to inhibition of gastric motility and increase in bile reflux via activation of peripheral corticotropin-releasing factor 2, 5-hydroxytriptamine 3, and cholecystokinin 1 receptors with central dopamine 2 receptor. Our study predicts that gastroparesis induced by antimotility drugs, stress, functional dyspepsia, Parkinson disease, diabetes mellitus, and other conditions worsens, and gastroprokinetic agents prevent the severity of nonsteroidal anti-inflammatory drugs-induced gastric antral ulcers.

Acute, high-dose metformin (MET, 2 mM) results in partial complex I inhibition in ischemia (ISC)-modified mitochondria. Mitochondrial permeability transition pore (MPTP) opening increases cardiac injury during ISC-reperfusion (REP). We evaluated whether MET (2 mM) can decrease MPTP opening in aged hearts during REP. Sestrin2 (Sesn2) regulates metabolism through activation of AMP-dependent protein kinase. Sesn2 decreases in aged hearts. The knockout (KO) of Sesn2 mimics the aging phenotype. Inactivation of glycogen synthase kinase-3 β (GSK-3β) via serine-9 phosphorylation decreases MPTP opening. We assessed if 2 mM MET given during early REP can decrease cardiac injury by partial blockade of complex I with decreased MPTP opening and if the protection depends on Sesn2-mediated GSK-3β phosphorylation. C57BL/6BJ male mice (22-24 months) and adult Sesn2 KO mice were evaluated. MET dose-dependently inhibited NADH oxidase activity in permeabilized mitochondria in both aged and Sesn2 KO greater after 25 minutes of ISC. MET (2 mM) given during REP decreased infarct size in aged hearts. MET improved calcium retention capacity in both aged wild-type and adult Sesn2 KO mice. MET treatment only increased phosphorylation of GSK-3β in aged heart mitochondria but not in Sesn2 KO hearts. Thus, high-dose MET at REP partially inhibits complex I and decreases MPTP opening. The decreased MPTP susceptibility downstream of complex I inhibition is not fully dependent on GSK-3β inhibition. Complex I downregulation with acute, high-dose MET has translational potential to protect the aged heart. SIGNIFICANCE STATEMENT: This study explores the efficacy and mechanism of acute high-dose metformin treatment in reducing mitochondrial-driven cardiac injury during reperfusion after stop-flow ischemia in the high-risk aged heart. Metformin dose-dependently inhibits complex I (NADH oxidation) in ischemia-altered mitochondria. Metformin given during early reperfusion mitigated MPTP opening as the mechanism of decreased reperfusion injury. Thus, modulation of complex I via metformin at reperfusion has potential translational application to mitigate injury during ST-elevation myocardial infarction in the high-risk aged heart.