Pharmacological Concentrations Research Articles

Metformin restores autophagic flux and mitochondrial function in late passage myoblast to impede age-related muscle loss

Sarcopenia, which refers to age-related muscle loss, presents a significant challenge for the aging population. Age-related changes that contribute to sarcopenia include cellular senescence, decreased muscle stem cell number and regenerative capacity, impaired autophagy, and mitochondrial dysfunction. Metformin, an anti-diabetic agent, activates AMP-activated protein kinase (AMPK) and affects various cellular processes in addition to reducing hepatic gluconeogenesis, lowering blood glucose levels, and improving insulin resistance. However, its effect on skeletal muscle cells remains unclear. This study aimed to investigate the effects of metformin on age-related muscle loss using a late passage C2C12 cell model. The results demonstrated that metformin alleviated hallmarks of cellular senescence, including SA-β-gal activity and p21 overexpression. Moreover, treatment with pharmacological concentrations of metformin restored the reduced differentiation capacity in late passage cells, evident through increased myotube formation ability and enhanced expression of myogenic differentiation markers such as MyoD, MyoG, and MHC. These effects of metformin were attributed to enhanced autophagic activity, normalization of mitochondrial membrane potential, and improved mitochondrial respiratory capacity. These results suggest that pharmacological concentrations of metformin alleviate the hallmarks of cellular senescence, restore differentiation capacity, and improve autophagic flux and mitochondrial function. These findings support the potential use of metformin for the treatment of sarcopenia.

Glucagon Stimulation Test and Insulin Secretory Capacity in Clinical Assessment of Incretin-Based Therapy for Diabetes.

Evaluation of insulin secretory capacity is essential to understand the pathophysiological condition of individuals with diabetes and to assess the efficacy of drugs used in treatment of the disease. The 1 mg intravenous glucagon stimulation test (GST) is widely used to evaluate residual β cell function; we previously reported that GST assessment of insulin secretory capacity is useful in assessing the efficacy of glucagon-like peptide-1 receptor agonists (GLP-1RAs). However, recent reports indicate that pharmacological concentrations of glucagon stimulate insulin secretion through GLP-1 receptors, confounding the issue. The present studies were undertaken to reassess the reliability of the GST for evaluation of insulin secretory capacity under GLP-1RAs and dipeptidyl peptidase-4 inhibitors (DPP-4is). Our first study comprised individuals initiated with the treatment of GLP-1RAs evaluated by GSTs before and after treatment. Although the fasting C-peptide levels (CPR) were elevated after treatment, the induction of insulin secretion by glucagon was significantly reduced. Our second study compared glucagon-induced insulin secretion between DPP-4i users and non-users, assessed by GST after propensity score matching. While the fasting CPR were similar in the two investigations, glucagon-induced insulin secretion was significantly lower with DPP-4i use. These results suggest that GST might underestimate insulin secretory capacity under incretin-based therapy.

The Beneficial Effects of Prenatal Biotin Supplementation in a Rat Model of Intrauterine Caloric Restriction to Prevent Cardiometabolic Risk in Adult Female Offspring.

Numerous studies indicate that intrauterine growth restriction (IUGR) can predispose individuals to metabolic syndrome (MetS) in adulthood. Several reports have demonstrated that pharmacological concentrations of biotin have therapeutic effects on MetS. The present study investigated the beneficial effects of prenatal biotin supplementation in a rat model of intrauterine caloric restriction to prevent cardiometabolic risk in adult female offspring fed fructose after weaning. Female rats were exposed to a control (C) diet or global caloric restriction (20%) (GCR), with biotin (GCRB) supplementation (2 mg/kg) during pregnancy. Female offspring were exposed to 20% fructose (F) in drinking water for 16 weeks after weaning (C, C/F, GCR/F, and GCRB/F). The study assessed various metabolic parameters including Lee's index, body weight, feed conversion ratio, caloric intake, glucose tolerance, insulin resistance, lipid profile, hepatic triglycerides, blood pressure, and arterial vasoconstriction. Results showed that GCR and GCRB dams had reduced weights compared to C dams. Offspring of GCRB/F and GCR/F dams had lower body weight and Lee's index than C/F offspring. Maternal biotin supplementation in the GCRB/F group significantly mitigated the adverse effects of fructose intake, including hypertriglyceridemia, hypercholesterolemia, hepatic steatosis, glucose and insulin resistance, hypertension, and arterial hyperresponsiveness. This study concludes that prenatal biotin supplementation can protect against cardiometabolic risk in adult female offspring exposed to postnatal fructose, highlighting its potential therapeutic benefits.

Targeting the redox vulnerability of acute myeloid leukaemia cells with a combination of auranofin and vitamin C.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by complex molecular and cytogenetic abnormalities. Pro-oxidant cellular redox status is a common hallmark of AML cells, providing a rationale for redox-based anticancer strategy. We previously discovered that auranofin (AUF), initially used for the treatment of rheumatoid arthritis and repositioned for its anticancer activity, can synergize with a pharmacological concentration of vitamin C (VC) against breast cancer cell line models. In this study, we observed that this drug combination synergistically and efficiently killed cells of leukaemic cell lines established from different myeloid subtypes. In addition to an induced elevation of reactive oxygen species and ATP depletion, a rapid dephosphorylation of 4E-BP1 and p70S6K, together with a strong inhibition of protein synthesis were early events in response to AUF/VC treatment, suggesting their implication in AUF/VC-induced cytotoxicity. Importantly, a study on 22 primary AML specimens from various AML subtypes showed that AUF/VC combinations at pharmacologically achievable concentrations were effective to eradicate primary leukaemic CD34+ cells from the majority of these samples, while being less toxic to normal cord blood CD34+ cells. Our findings indicate that targeting the redox vulnerability of AML with AUF/VC combinations could present a potential anti-AML therapeutic approach.

High-Dose Intravenous Vitamin C Combined with Docetaxel in Men with Metastatic Castration-Resistant Prostate Cancer: A Randomized Placebo-Controlled Phase II Trial.

This is the first randomized, placebo-controlled, double-blind trial to evaluate HDIVC in cancer treatment. The addition of HDIVC to docetaxel in patients with mCRPC does not improve PSA response, toxicity, or other clinical outcomes compared with docetaxel alone. The routine use of HDIVC in mCRPC treatment is not supported outside of clinical trials.

Inositol Restores Appropriate Steroidogenesis in PCOS Ovaries Both In Vitro and In Vivo Experimental Mouse Models.

Androgen excess is a key feature of several clinical phenotypes of polycystic ovary syndrome (PCOS). However, the presence of FSH receptor (FSHR) and aromatase (CYP19A1) activity responses to physiological endocrine stimuli play a critical role in the pathogenesis of PCOS. Preliminary data suggest that myo-Inositol (myo-Ins) and D-Chiro-Inositol (D-Chiro-Ins) may reactivate CYP19A1 activity. We investigated the steroidogenic pathway of Theca (TCs) and Granulosa cells (GCs) in an experimental model of murine PCOS induced in CD1 mice exposed for 10 weeks to a continuous light regimen. The effect of treatment with different combinations of myo-Ins and D-Chiro-Ins on the expression of Fshr, androgenic, and estrogenic enzymes was analyzed by real-time PCR in isolated TCs and GCs and in ovaries isolated from healthy and PCOS mice. Myo-Ins and D-Chiro-Ins, at a ratio of 40:1 at pharmacological and physiological concentrations, positively modulate the steroidogenic activity of TCs and the expression of Cyp19a1 and Fshr in GCs. Moreover, in vivo, inositols (40:1 ratio) significantly increase Cyp19a1 and Fshr. These changes in gene expression are mirrored by modifications in hormone levels in the serum of treated animals. Myo-Ins and D-Chiro-Ins in the 40:1 formula efficiently rescued PCOS features by up-regulating aromatase and FSHR levels while down-regulating androgen excesses produced by TCs.

Empagliflozin, an SGLT2i, relaxes coronary smooth muscle at pharmacological concentrations

Morbidity and mortality due to heart failure with preserved ejection fraction (HFpEF) is increasing and there are no universally recognized strategies for treatment. Sodium-glucose co-transporter type 2 inhibitors (SGLT2i), such as Empagliflozin, have been shown to reduce myocardial stiffness and improve coronary microvascular performance, but mechanisms are unclear. Previous studies in rabbit aorta and rat mesentery suggest Empagliflozin relaxes smooth muscle through activation of K+ channels. This study investigated the effects of therapeutic and pharmacological concentrations of Empagliflozin (10-300 μM) on the isometric tension of left circumflex (CFX) coronary arteries from Yorkshire swine. We tested the hypothesis that Empagliflozin would relax coronary smooth muscle via a concentration-dependent manner, involving K+ channels. CFX arteries were dissected, cleaned of adventitia, cut into 3 mm rings (n = 16), and mounted in organ baths containing Krebs-Henseleit buffer (37 °C) for isometric tension studies. Each CFX segment was adjusted to optimal preload (~3 g passive tension) as determined by <10 % change in active tension in response to 60 mM KCl. In order to determine whether Empagliflozin caused relaxation and, if so, by what mechanism, rings were contracted with either 30 mM K+ or the thromboxane mimetic U46619 (1 μM). Once tension plateaued, Empagliflozin (10-300 μM) was added in increasing concentrations. Therapeutic concentrations of Empagliflozin (10-30 μM), elicited no significant relaxation. Pharmacologic concentrations of Empagliflozin (≥100 μM) caused significant relaxation from ~15 ± 11% to ~45 ± 10% in response to 100 μM vs. 300 μM Empagliflozin, respectively. Relaxation was not different between rings contracted with K+ or U46619, suggesting membrane hyperpolarization via K+ channels is not involved. Our results represent the first study of the effects of Empagliflozin on coronary smooth muscle reactivity. Data support the idea that relaxation of coronary smooth muscle by Empagliflozin occurs at supra-pharmacologic concentrations in vitro, but K+ channel activation appears to be an unlikely mechanism. This project was supported by National Institutes of Health grant R01 HL158723. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Melatonin facts: Melatonin lacks immuno-inflammation boosting capacities at the molecular and cellular levels

Among the properties melatonin is claimed to possess, are the immuno-inflammation inductive capacities that would be responsible of some of the paramount of activities melatonin is reported to have in most of the human pathological conditions. In the present paper, we measured the effect of melatonin on established cellular models of immuno-inflammation, and found none. The discrepancies are discussed, especially because those properties are reported at pharmacological concentration (1 μM and beyond) at which the melatonin receptors are desensitized by internalization, leading to putative non-receptor-dependent mechanism of action.

Riboflavin protects against pancreatic cancer metastasis by targeting TGF-β receptor 1

The inhibition of transforming growth factor-β1 (TGF-β1) signaling by targeting TGF-β receptor 1 (TβR1) has been considered as an ideal approach for the prevention of pancreatic cancer metastasis. Utilizing a pharmacophore model for TβR1 inhibitors, candidate compounds with the potential TβR1 binding ability were screened from the U.S. Food and Drug Administration (FDA) database, and riboflavin (RF) with a highest fit value was chosen to investigate its binding ability to TβR1 and effect on TGF-β1 signaling in pancreatic cancer cells. Molecular docking and cellular thermal shift assay (CETSA) proved that RF at pharmacological concentrations could directly bind to TβR1. Further studies showed that pharmacological concentrations of RF in vitro could block TGF-β1 signaling, suppress the migration and invasion, and prevent epithelial-mesenchymal transition (EMT) process of pancreatic cancer cells in the absence or presence of TGF-β1 stimulation, indicating that RF presented anti-metastatic effect in pancreatic cancer cells. Knockdown of TβR1 could significantly attenuate the effects of RF on the migration and EMT process in pancreatic cancer cells, further confirming that the anti-metastatic effect of RF was achieved by blocking TGF-β1 signaling after binding to TβR1. Moreover, in a mouse model of pancreatic cancer metastasis, it was certified that RF administration could block lung and liver metastases, TGF-β1 signaling and EMT process of pancreatic cancer in vivo. In summary, our findings showed that RF could block TGF-β1 signaling by directly binding to TβR1, thereby suppressing the metastasis of pancreatic cancer cells by inhibiting EMT process both in vitro and in vivo.

PEGylated Chitosan Biodegradable Nanoparticles Delivery of Salvia officinalis and Melissa officinalis for Enhanced Brain Targeting

Background: Alzheimer's disease (AD) is a progressive neurodegenerative condition characterized by the gradual decline of cognitive abilities, primarily caused by impairments in the cholinergic system. AD is diagnosed based on the presence of specific pathological features, in-cluding senile plaques, neurofibrillary tangles, and the loss of neurons and synapses. Despite on-going efforts, the etiology of AD remains unclear, and there is a significant lack of effective treatments to meet the medical needs of affected individuals. The complex nature of AD, involv-ing multiple factors, presents challenges in the development of potential therapies. Numerous ob-stacles hinder the achievement of optimal pharmacological concentration of promising molecules for AD treatment. These obstacles include the presence of the blood-brain barrier (BBB), which restricts the entry of therapeutic agents into the brain, as well as issues related to poor bioavaila-bility and unfavorable pharmacokinetic profiles. Unfortunately, many therapeutically promising compounds have failed to overcome these hurdles and demonstrate efficacy in treating AD. Methods: The PEGylated chitosan nanoconjugate was developed and evaluated for delivery of anti-Alzheimer natural extract of Salvia officinalis and Melissa officinalis to the brain. The nano-conjugates (S-PCN and M-PCN) were developed by ionic gelation technique. Result: The nanoconjugates (S-PCN and M-PCN) were evaluated for various optical and in-vitro parameters. MTT assay on UCSD229i-SAD1-1 human astrocytoma cells indicated IC50 values of 0.42, 0.49, 0.67, and 0.75 μM for S-PCN, M-PCN formulations, and free Salvia officinalis and Melissa officinalis extracts, respectively. The In vitro assessments using cell lines have confirmed the improved uptake and distribution of nanoconjugates compared to free extracts. These findings were validated through confocal microscopy and apoptosis assays, revealing a substantial in-crease in the accumulation of nanoconjugates within the brain. The targeting potential OF M- PCN over S-PCN was found to be 2-fold significant. Conclusion: Based on the findings, it can be inferred that biodegradable PEGylated chitosan nanoconjugates hold promise as effective nano-targeting agents for delivering anti-Alzheimer drugs to the brain. The incorporation of PEGylated chitosan nanoparticles in this approach demonstrates enhanced delivery capabilities, ultimately leading to improved therapeutic out-comes.

Diallyl Trisulfide Induces ROS-Mediated Mitotic Arrest and Apoptosis and Inhibits HNSCC Tumor Growth and Cancer Stemness.

Despite advances in therapeutic approaches, the five-year survival rate for head and neck squamous cell carcinoma (HNSCC) patients is still less than fifty percent. Research has indicated that the consumption of Allium vegetables or processed garlic containing diallyl trisulfide (DATS) can lower the risk of multiple types of cancer. Nevertheless, the effectiveness and underlying mechanisms of DATS against HNSCC have not been thoroughly explored until the current study. In this research, it was found that DATS notably curtailed the growth and viability of HNSCC cells. Additionally, DATS triggered a significant G2/M cell cycle arrest in these cells, accumulating cyclin B1, Cip1/p21, and Ser-10 phospho-histone H3-this was indicative of mitotic arrest attenuated by NAC pretreatment, suggesting the role of reactive oxygen species (ROS) induction. The production of ROS induced by DATS led to DNA damage and apoptosis, a process associated with elevated levels of cleaved caspase-3 and cleaved PARP, along with reduced XIAP. When HNSCC cells were exposed to pharmacological concentrations of DATS, it resulted in the suppression of cancer stem cell (CSC) populations, as indicated by a decrease in the CD133high/CD44high cell fraction, reduced aldehyde dehydrogenase 1 (ALDH1) activity, inhibited spheroid formation and downregulated SOX2 and Oct4 expression. Furthermore, the administration of DATS to tumor xenografts demonstrated its in vivo capacity to hinder CSCs. Further, DATS treatment inhibited the growth of UMSCC-22B head and neck cancer tumor xenograft in immunocompromised mice. Overall, DATS inhibited cell proliferation; induced cell cycle mitotic arrest and apoptosis involving DNA damage through ROS generation; reduced the CSC fraction and spheroid formation; and downregulated SOX2 and Oct4 expression. More importantly, DATS inhibited HNSCC tumor growth and CSC fraction in vivo. Thus, DATS could be a potential anticancer agent that can be used against head and neck cancer.

Daphnia magna an emerging environmental model of neuro and cardiotoxicity of illicit drugs.

Cocaine, methamphetamine, ectasy (3,4-methylenedioxy amphetamine (MDMA)) and ketamine are among the most consumed drugs worldwide causing cognitive, oxidative stress and cardiovascular problems in humans. Residue levels of these drugs and their transformation products may still enter the aquatic environment, where concentrations up to hundreds of ng/L have been measured. In the present work we tested the hypothesis that psychotropic effects and the mode of action of these drugs in D. magna cognitive, oxidative stress and cardiovascular responses are equivalent to those reported in humans and other vertebrate models. Accordingly we expose D. magna juveniles to pharmacological and environmental relevant concentrations. The study was complemented with the measurement of the main neurotransmitters involved in the known mechanisms of action of these drugs in mammals and physiological relevant amino acids. Behavioural cognitive patters clearly differentiate the 3 psychostimulant drugs (methamphetamine, cocaine, MDMA) from the dissociative one ketamine. Psychostimulant drugs at pharmacological doses (10–200 μM), increased basal locomotion activities and responses to light, and decreased habituation to it. Ketamine only increased habituation to light. The four drugs enhanced the production of reactive oxygen species in a concentration related manner, and at moderate concentrations (10–60 μM) increased heartbeats, diminishing them at high doses (200 μM). In chronic exposures to environmental low concentrations (10–1000 ng/L) the four drugs did not affect any of the behavioural responses measured but methamphetamine and cocaine inhibited reproduction at 10 ng/L. Observed effects on neurotransmitters and related metabolites were in concern with reported responses in mammalian and other vertebrate models: cocaine and MDMA enhanced dopamine and serotonin levels, respectively, methamphetamine and MDMA decreased dopamine and octopamine, and all but MDMA decreased 3 MT levels. Drug effects on the concentration of up to 10 amino acids evidence disruptive effects on neurotransmitter synthesis, the urea cycle, lipid metabolism and cardiac function.

Dabigatran pharmacokinetic-pharmacodynamic in sheep: Informing dose for anticoagulation during cardiopulmonary bypass.

The effect of the anticoagulant, dabigatran, and its antagonist, idarucizumab, on coagulation remains poorly quantified. There are few pharmacokinetic-pharmacodynamic data available to determine dabigatran dose in humans or animals undergoing cardiopulmonary bypass. Five sheep were given intravenous dabigatran 4mg/kg. Blood samples were collected for thromboelastometric reaction time (R-time) and drug assay at 5, 15, 30, 60, 120, 240, 480min, and 24h. Plasma dabigatran concentrations and R-times were analyzed using an integrated pharmacokinetic-pharmacodynamic model using non-linear mixed effects. The impact of idarucizumab 15mg/kg administered 120min after dabigatran 4mg/kg and its effect on R-time was observed. A 2-compartment model described dabigatran pharmacokinetics with a clearance (CL 0.0453L/min/70kg), intercompartment clearance (Q 0.268L/min/70kg), central volume of distribution (V1 2.94L/70kg), peripheral volume of distribution (V2 9.51L/70kg). The effect compartment model estimates for a sigmoid EMAX model using Reaction time had an effect site concentration (Ce50 64.2mg/L) eliciting half of the maximal effect (EMAX 180min). The plasma-effect compartment equilibration half time (T1/2keo) was 1.04min. Idarucizumab 15mg/kg reduced R-time by approximately 5min. Dabigatran reversibly binds to the active site on the thrombin molecule, preventing activation of coagulation factors. The pharmacologic target concentration strategy uses pharmacokinetic-pharmacodynamic information to inform dose. A loading dose of dabigatran 0.25mg/kg followed by a maintenance infusion of dabigatran 0.0175mg/kg/min for 30min and a subsequent infusion dabigatran 0.0075mg/kg/min achieves a steady state target concentration of 5mg/L in a sheep model.

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Partner Gene Associated Trinary Fusion and Orchestration of Multiple Molecular Events in Acute Promyelocytic Leukemia with Variant Rara Fusion Genes

Background All-trans retinoic acid (ATRA) has been used with great success in acute promyelocytic leukemia (APL) cases with PML::RARA fusion gene (FG). There are still PML::RARA negative cases manifest as APL. So far, 19 RARA fusion partner genes have been reported, with all reported as 5′ partners ( X::RARA). Intriguingly, a significant proportion of X::RARA cases were resistant to ATRA, and mechanisms are yet to be elucidated. Here we report the finding of a novel type of trinary fusion and the orchestrating of multiple molecular events that confer leukemogenesis and ATRA resistance in X::RARA-APL. Methods A total of 23 cases (Fig. 1A) with RNA-seq and WGS data from 10 centers were enrolled. We used Arriba for routine FG calling and manually investigated the nonconventional fusion sequence. RT-PCR, Sanger sequencing, and optical genome mapping (OGM) were used to validate the FGs and to determine whether the RARA 5′ and 3′ fusion events were located on the same cistron. The responsiveness of the fusion proteins to ATRA was evaluated using an optimized UAS/GAL4 reporter system. Results Among the cases enrolled, there were 17 males and 6 females, aged 3-66 (median 32) years. Morphological and immunophenotype analyses all showed features of APL. We first identified the STAT3::RARA fusion event in an ATRA-resistant case (the index case, #A1). Moreover, we noticed another fusion event of truncated RARA exon 9 and a 3′ partner sequence that did not map to any annotated protein-coding genes. Further analysis determined it was the RC sequence of the transposable element (TE) LTR40a with the annotated loci at 17p11.2. Both OGM (at the genome level) and RT-PCR (Fig. 1A) confirmed that the two RARA fusion events were in the same cistron. Thus, the fusion transcript being STAT3::RARA::LTR40a-RC spliced in tandem, but not separate transcripts of STAT3::RARA and RARA::LTR40a-RC. We named this novel form of tandem splicingtrinary fusion. We, therefore, recruit X::RARA-APL cases to investigate the recurrence of RARA 3′ fusion. We identified 9 RARA 5′ partner genes in 23 cases (Fig. 1B), with TTMV -ORF2, STAT5B, STAT3, IRF2BP1, THRAP3, and TNRC18 in multiple cases. Ten (43.5%) cases also harbor RARA 3′ fusion partners. The partner of case #A1_8 were TEs, and the partner of case #A9_10 was the same gene as their 5′ one. Thirteen cases did not harbor 3′ fusions. Notably, both TNRC18::RARA cases (#15_16) carry mutations on their RARA ligand binding domain (LBD) at the primary onset. Gene expression analysis confirmed the aberrant activation of the involved TEs. All TEs involved in the RARA 3' fusions confer a poly_A signal sequence, which was essential for a mRNA. In-depth analysis indicated that TEs participate in forming RARA-FGs through a transposition mechanism rather than a translocation mechanism. The molecular events addition to RARA 5′ splicing showed a 5′ partner associated pattern: all of the 4 STAT3::RARA and 4 STAT5B::RARA cases harbor TEs as their 3′ partner, both TNRC18::RARA cases carry LBD mutation, while all 7 TTMV -ORF2::RARA cases did not harbor 3′ splicing or LBD mutation. Remarkably, although the RARA 3' splicing sites varied, they all resulted in partial or complete exon 9 truncation. RARA exon 9 encodes helix 11_12 (H11_12) of its LBD, which is pivotal in allosteric response to ATRA. The RARA trinary fusion will result in LBD-H12 or H11_12 castration due to the 3' splicing, then lead to losing responsiveness to ATRA via an allosteric disability mechanism (Fig. 1C, 1D). Experiments confirmed that cells transfected with STAT3::RARA fusion with intact LBD responded well to ATRA, while both the fusion in case #A1 and artificial fusion with LBD-H12 castration showed no response to physiological and pharmacological concentrations of ATRA (Fig. 1E). Conclusions This is the first report of pathological trinary fusion, the TEs and transposition mechanism participating in composing pathological fusion genes, and the RARA LBD castration confers ATRA resistance in X::RARA-APL. Also, there might be another synergistic mechanism of RARA 5' fusion and LBD mutation. The formation of trinary fusion and gain of LBD mutation, in addition to X::RARA fusion, requires more genome splicing or transposition or mutation events, which explains the rarity of X::RARA-APL. This study highlights mechanisms of partner gene-associated trinary fusion and the orchestrating of multiple molecular events in the leukemogenesis in X::RARA-APL cases.

Trinary Fusion with Ligand Binding Domain Castration Is Crucial in Rarg-Driven APL and Render Unresponsive to ATRA Via Allosteric Disability Mechanism

Introduction All-trans retinoic acid (ATRA) has been used with great success in acute promyelocytic leukemia (APL) cases with PML::RARA fusion gene (FG). There are still PML::RARA negative cases that manifest as APL, and they often carry FGs involved in RARA, RARB, or RARG. X::RARG positive APL ( RARG-APL) cases have been increasingly reported in recent years, thanks to the utilization of transcriptome sequencing (RNA-seq) and whole genome sequencing (WGS). Almost all evaluable RARG-APL cases were resistant to ATRA, and no mechanism has been elucidated. Intriguingly, cells transformed by artificial X::RARG were extremely sensitive to ATRA (PMID: 25510432), indicating intricate mechanisms unrevealed. This study first and systematically unveils the distinctive features and pivotal molecular mechanism of RARG-driven APL. Methods A total of 22 RARG-APL cases with RNA-seq and WGS data from 11 centers were enrolled (Fig. 1A). We used Arriba for routine FG calling and manually investigated the non-coding fusion sequence. Reverse transcription PCR (RT-PCR) and Sanger sequencing were used to validate the FGs and to determine whether the RARG 5′ and 3′ fusion events were located on the same cistron. The responsiveness of the fusion proteins to ATRA was evaluated using an optimized UAS/GAL4 reporter system. Results Among the cases enrolled, there were 15 males and 7 females, aged 0.9-69 (median 38) years. Morphological and immunophenotype analyses all showed features of APL. Routine FGs calling reported RARG fusion to a 5′ partner in each case, with variable RARG splicing sites (Fig. 1A). There were 7 RARG 5' partner genes, with CPSF6, HNRNPC, and NUP98 observed in multiple cases. In the 3 index cases, we identified that they all had RARG 3‘ fusion events besides the 5‘ one (Fig. 1A). RT-PCR confirmed that the 5′ and 3′ RARG fusion events were in the same cistron in each case. Such as, the fusion transcript is the insertion of RARG-e4_9 (e refers to exon) between NPM1-e4 and e11 in case #G1, rather than two separate transcripts of NPM1-e4:: RARG-e4 and RARG-9:: NPM1-e11. We named this novel form of tandem splicingtrinary fusion. Analysis of the 19 validation cases further confirmed that all RARG-APL cases had RARG 3′ fusion events (Fig. 1A). We also validated that the 5′ and 3′ RARG fusion events were in the same cistron in 3 more cases, of which archived cDNA were available. Remarkably, the 3' splicing sits of all cases were consistently at the end of RARG-e9, leading to RARG-e10 truncation. The RARG 3' partner gene was the same as its 5' partner in 11 cases. But in the other 11 cases, the RARG 3' partner was a transposon element (TE) sequence, the most common being a LINE-L2a (8 cases). Sequences analysis indicated that the committed locus of the involved LINE-L2a was at 4.2 Kbp upstream of the RARG gene. Gene expression analysis confirmed the aberrant activation of the involved TEs. All TEs involved in the RARG 3' fusions confer a poly_A signal sequence to the fusion transcript, which was essential for a mRNA. In-depth analysis indicated that TEs participate in the formation of RARG-FGs through a transposition mechanism rather than a translocation mechanism. RARG-e10 encodes helix 11_12 (H11_12) of its ligand binding domain (LBD), which plays a pivotal role in allosteric response to ATRA (PMID: 31178221). The trinary fusion protein with RARG LBD-H11_12 castration will lose responsiveness to ATRA via an allosteric disability mechanism (Fig. 1B, 1C). Experiments confirmed that cells transfected with X::RARG fusion with intact LBD responded well to ATRA, while fusion with LBD-H11_12 castration showed no response to physiological and pharmacological concentrations of ATRA (Fig. 1D, 1E). Conclusions This study clearly revealed that trinary fusion and LBD castration are crucial molecular etiologies in RARG-APL. The trinary fusion orchestrates the aberrant activation of RARG (5‘ splicing) and the complete allosteric disability in responding to ATRA (3‘ splicing). Thus, this study elegantly explains the mechanism of leukemogenesis and the extensive ATRA resistance of RARG-APL. The formation of trinary fusion requires two steps of genome splicing or transposition events, which explains the rarity of RARG-APL. This also clarifies that RARG-APL has a significant disparity in molecular mechanism from PML::RARA-positive APL, and we suggest that it should be considered as a separate entity of acute myeloid leukemia.

Fatty Acid Binding Protein FABP5 Inhibition Activates Retinoic Acid Signaling and Induces Differentiation in Acute Myeloid Leukemia

Introduction. Accumulation of undifferentiated immature blood cells is a hallmark of myeloid neoplasia (MN), and indicates decoupling of terminal-differentiation from proliferation. Therefore, reinstating to a programmed state of differentiation (lineage-maturation) is a treatment approach, exemplified by all-trans retinoic acid (ATRA) treatment of acute promyelocytic leukemias that contain t(15;17) (q24;q21). This translocation inactivates retinoic acid receptor alpha (RARA) due to its fusion with promyelocytic leukemia PML resulting in PML-RARA. This fusion gene responsible for cellular transformation confers sensitivity to treatment with ATRA. However, ATRA's therapeutic-success in APLs has not extended to other AML phenotypes. We and others have shown that RA is a ligand, not just for RARs, but also pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a member of the nuclear hormone receptor superfamily transcription factors that regulate several metabolic pathways. RA is transported by two carrier proteins, cellular retinoid-binding protein CRABP2, or fatty-acid-binding protein 5 (FABP5), depending on cellular context. These carriers preferentially deliver RA to RARs versus PPARβ/δ respectively. Somatic lesions such as loss of function TET2 or Tp53 mutation, NPM1 and ASXL1 mutations, are associated with upregulated FABP5 and downregulated CRABP2. In AML cells with these mutations, therefore, the high FABP5 can be expected to deliver RA to PPARβ/δ instead of RAR, to potentially contribute to decoupling of differentiation from proliferation. We therefore investigated this possibility and its treatment implications. Methods. Overexpression, gene knockdown/knockout (ko/kd) of key regulators of RAR and PPARβ/δ were generated in specific genotypes of AMLs. Small molecule inhibitors of FABP5 were used to further interrogate the pathways in a translationally relevant manner, both in vitro, in both malignant and normal cells, and in pre-clinical in vivo models. Results. Gene expression analysis of AMLs in the Beat AML and TCGA databases demonstrated upregulation of pro-survival PPARβ/δ target genes including FABP5 and downregulation of differentiation promoting RAR pathway genes including CRABP2in AMLs 1. In particular, FABP5 was highly expressed in AMLs containing TET2 NPM1, TP53 and ASXL1 mutations. AML cell lines OCIAML3 (NPM1 ins/DNMT3a R882c), OCIAML5 (ASXL1 Y591*/TET2 S825*), THP1 (TP53 del) and isogenic CRISPR engineered K562 (TET2 +/+/TET2 -/-) demonstrated 4-100 fold higher FABP5 expression compared to normal myeloid precursors. These models did not differentiate in response to pharmacologic concentrations of ATRA. To test if FABP5 is necessary and sufficient to maintain the undifferentiated state of these cells in the presence of ATRA, we genetically inactivated FABP5 either using RNAi (kd) or CRISPR (ko). FABP5 knockdown or knockout cells created sensitivity to ATRA, resulting in terminal-differentiation as observed by flow cytometry for terminal-differentiation markers ( See Figure). FABP5 was then inhibited using highly specific small molecule FABP5 inhibitors with a 56-fold higher affinity compared to ATRA 1: combination of iFABP5 and ATRA induced terminal-differentiation with significant upregulation of the monocyte lineage markers CD14 and granulocyte lineage marker CD11b measured by flow cytometry at 72h. Toinvestigatewhether knocking down FABP5 and ATRA treatment affects gene expression changes we performed transcriptomic profiling of OCIMAL3 Scr, shFABP5 cells treated with or without ATRA using whole exome RNA sequencing. Treatment of FABP5 kd OCIMAL3 cells with ATRA resulted in the upregulation of RAR pathways genes and downregulation of proliferation promoting genes. Conclusion. In sum, recoupling of lineage-maturation to proliferation can be achieved in non-APL AMLs that highly express the RA carrier FABP5, by inhibiting FABP5 using small molecules. This candidate treatment modality is distinct from cytotoxic chemotherapy, and importantly, can spare normal hematopoiesis.

235 Interactive Effects of Zinc Oxide and Mannan Oligosaccharides on Nursery Swine Performance

Abstract Peer-reviewed data have suggested that Mannan oligosaccharide [Actigen (ACT)] could positively influence gut barrier function and limit pathogenic bacteria adhesion to epithelial cells, thus representing a feasible alternative or a complement to pharmacological concentrations of zinc oxide (ZnO) in nursery swine diets. To scrutinize this hypothesis, a total of 2,592 pigs (initial BW 6.9 ± 0.4 kg) were used in a 37-day trial to determine the interactive effects of ZnO and ACT on nursery pig performance. Pens of 36 mixed sex pigs were randomly assigned to 1 of 6 dietary treatments with 12 replications per treatment. Treatments were arranged in a 2 × 3 factorial arrangement with main effects of zinc (100 or 3,000 ppm) and ACT (0, 400, and 800 ppm). Diets were fed in two phases: phase 1 from d 0 to d 21 and phase 2 from d 22 to d 37, which represented the experimental and common diets, respectively. No significant ZnO × ACT interactions (P > 0.10) were observed in growth performance or dead and removals in this study (Table 1). From d 0 to d 21, pigs fed diets with 3,000 ppm Zn had greater (P < 0.0001) ADFI, greater (P < 0.0001) ADG, and improved (P < 0.0001) feed efficiency compared with pigs fed diets containing 100 ppm Zn. From d 22 to d 37, pigs initially fed diets with 3,000 ppm Zn had reduced (P = 0.009) ADG and poorer (P = 0.0009) G:F, without evidence for differences (P = 0.615) in ADFI, compared with pigs fed diets containing 100 ppm of Zn. Additionally, pigs fed diets containing 3,000 ppm of Zn marginally increased (P = 0.077) removals prevalence compared with pigs fed diets with less Zn concentration. However, pigs fed diets with 3,000 ppm Zn and incremental levels of ACT marginally decreased (linear, P = 0.083) dead and removals prevalence. In conclusion, pigs fed pharmacological ZnO concentration increased overall growth performance, yet it was drastically worsened when Zn was reduced to basal levels, despite ACT supplementation. Additionally, whereas pigs fed the greatest Zn level marginally increased morbidity occurrence, ACT marginally reduced morbidity and mortality.

Melatonin and Its Metabolites Can Serve as Agonists on the Aryl Hydrocarbon Receptor and Peroxisome Proliferator-Activated Receptor Gamma.

Melatonin is widely present in Nature. It has pleiotropic activities, in part mediated by interactions with high-affinity G-protein-coupled melatonin type 1 and 2 (MT1 and MT2) receptors or under extreme conditions, e.g., ischemia/reperfusion. In pharmacological concentrations, it is given to counteract the massive damage caused by MT1- and MT2-independent mechanisms. The aryl hydrocarbon receptor (AhR) is a perfect candidate for mediating the latter effects because melatonin has structural similarity to its natural ligands, including tryptophan metabolites and indolic compounds. Using a cell-based Human AhR Reporter Assay System, we demonstrated that melatonin and its indolic and kynuric metabolites act as agonists on the AhR with EC50's between 10-4 and 10-6 M. This was further validated via the stimulation of the transcriptional activation of the CYP1A1 promoter. Furthermore, melatonin and its metabolites stimulated AhR translocation from the cytoplasm to the nucleus in human keratinocytes, as demonstrated by ImageStream II cytometry and Western blot (WB) analyses of cytoplasmic and nuclear fractions of human keratinocytes. These functional analyses are supported by in silico analyses. We also investigated the peroxisome proliferator-activated receptor (PPAR)γ as a potential target for melatonin and metabolites bioregulation. The binding studies using a TR-TFRET kit to assay the interaction of the ligand with the ligand-binding domain (LBD) of the PPARγ showed agonistic activities of melatonin, 6-hydroxymelatonin and N-acetyl-N-formyl-5-methoxykynuramine with EC50's in the 10-4 M range showing significantly lower affinities that those of rosiglitazone, e.g., a 10-8 M range. These interactions were substantiated by stimulation of the luciferase activity of the construct containing PPARE by melatonin and its metabolites at 10-4 M. As confirmed by the functional assays, binding mode predictions using a homology model of the AhR and a crystal structure of the PPARγ suggest that melatonin and its metabolites, including 6-hydroxymelatonin, 5-methoxytryptamine and N-acetyl-N-formyl-5-methoxykynuramine, are excellent candidates to act on the AhR and PPARγ with docking scores comparable to their corresponding natural ligands. Melatonin and its metabolites were modeled into the same ligand-binding pockets (LBDs) as their natural ligands. Thus, functional assays supported by molecular modeling have shown that melatonin and its indolic and kynuric metabolites can act as agonists on the AhR and they can interact with the PPARγ at high concentrations. This provides a mechanistic explanation for previously reported cytoprotective actions of melatonin and its metabolites that require high local concentrations of the ligands to reduce cellular damage under elevated oxidative stress conditions. It also identifies these compounds as therapeutic agents to be used at pharmacological doses in the prevention or therapy of skin diseases.

Cell death induction and intracellular vesicle formation in human colorectal cancer cells treated with Δ9-Tetrahydrocannabinol

BackgroundΔ9-Tetrahydrocannabinol (Δ9-THC) is a principal psychoactive extract of Cannabis sativa and has been traditionally used as palliative medicine for neuropathic pain. Cannabidiol (CBD), an extract of hemp species, has recently attracted increased attention as a cancer treatment, but Δ9-THC is also requiring explored pharmacological application.ObjectiveThis study evaluated the pharmacological effects of Δ9-THC in two human colorectal cancer cell lines. We investigated whether Δ9-THC treatment induces cell death in human colorectal cancer cells.MethodsWe performed an MTT assay to determine the pharmacological concentration of Δ9-THC. Annxein V and Western blot analysis confirmed that Δ9-THC induced apoptosis in colorectal cancer cells. Metabolic activity was evaluated using MitoTracker staining and ATP determination. We investigated vesicle formation by Δ9-THC treatment using GW9662, known as a PPARγ inhibitor.ResultsThe MTT assay showed that treatment with 40 μM Δ9-THC and above inhibited the proliferation of colorectal cancer cells. Multiple intracytoplasmic vesicles were detected upon microscopic observation, and fluorescence-activated cell sorting analysis showed cell death via G1 arrest. Δ9-THC treatment increased the expression of cell death marker proteins, including p53, cleaved PARP-1, RIP1, and RIP3, suggesting that Δ9-THC induced the death of colorectal cancer cells. Δ9-THC treatment also reduced ATP production via changes in Bax and Bcl-2. Δ9-THC regulated intracytoplasmic vesicle formation by modulating the expression of PPARγ and clathrin, adding that antiproliferative activity of Δ9-THC was also affected.ConclusionIn conclusion, Δ9-THC regulated two functional mechanisms, intracellular vesicle formation and cell death. These findings can help to determine how cannabinoids can be used most effectively to improve the efficacy of cancer treatment.