Histone Demethylase JMJD2D Interacts With ß-Catenin to Induce Transcription and Activate Colorectal Cancer Cell Proliferation and Tumor Growth in Mice.

BACKGROUND & AIMS: Wnt signaling contributes to the development of colorectal cancer (CRC). We studied interactions between lysine demethylase 4D (KDM4D or JMJD2D) and ß-catenin, a mediator of Wnt signaling, in CRC cell lines and the effects on tumor formation in mice. METHODS: We obtained colorectal tumor specimens and surrounding nontumor colon tissues (controls) from patients undergoing surgery in China; levels of JMJD2D were measured by immunohistochemical or immunoblot analysis. JMJD2D expression was knocked down in CRC (CT26, HCT116, and SW480 cells) using small hairpin RNAs, and cells were analyzed with viability, flow cytometry, colony formation, and transwell migration and invasion assays. Cells were also grown as tumor xenografts in nude mice or injected into tail veins or spleens of mice, and metastases were measured. We performed promoter activity, co-immunoprecipitation, and chromatin immunoprecipitation assays. We also performed studies with Apcmin/+ and JMJD2D-knockout mice; these mice were crossed, and colorectal tumor formation in offspring (Apcmin/+Jmjd2d+/+ and Apcmin/+Jmjd2d-/-) was analyzed. JMJD2D-knockout and wild-type (control) mice were given azoxymethane followed by dextran sodium sulfate to induce colitis-associated CRC; some mice were given the JMJD2D inhibitor 5-chloro-8-hydroxyquinoline (5-c-8HQ) or vehicle to examine the effects of 5-c-8HQ on intestinal tumor formation. RESULTS: Levels of JMJD2D were significantly higher in human colorectal tumors than in control tissues and correlated with levels of proliferating cell nuclear antigen. JMJD2D knockdown reduced CRC cell proliferation, migration, and invasion, as well as growth of xenograft tumors and formation of metastases in mice. JMJD2D was required for expression of ß-catenin in CRC cell lines; ectopic expression of JMJD2D increased the promoter activities of genes regulated by ß-catenin (MYC, CCND1, MMP2, and MMP9). We found that JMJD2D and ß-catenin interacted physically and that JMJD2D demethylated H3K9me3 at promoters of ß-catenin target genes. JMJD2D-knockout mice developed fewer colitis-associated colorectal tumors than control mice, and their tumor tissues had lower levels of ß-catenin, MYC, cyclin D1, and proliferating cell nuclear antigen than tumors from control mice. Apcmin/+Jmjd2d-/- mice developed fewer and smaller colon tumors than Apcmin/+ mice. Mice given 5-c-8HQ developed smaller and fewer colitis-associated tumors, with lower levels of cell proliferation, than mice given vehicle. Apcmin/+ mice given 5-c-8HQ also developed fewer tumors in intestines and colons than mice given vehicle. CONCLUSIONS: Levels of the histone demethylase JMJD2D are increased in human colorectal tumors compared with nontumor colon tissues. JMJD2D interacts with ß-catenin to activate transcription of its target genes and promote CRC cell proliferation, migration, and invasion, as well as formation of colorectal tumors in mice.

Discovery of novel halogenated 8-hydroxyquinoline-based anti-MRSA agents: In vitro and QSAR studies.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has been considered to be one of global health problems due to limited classes of effective antimicrobial drugs. Herein, 8-hydroxyquinoline (8HQ) and its derivatives (1-7) were investigated for their anti-MRSA and antioxidant activities. Cloxyquin (2), a halogenated 8HQ, exerted the highest antimicrobial activity (MIC50 ≤ 5.57 µM) with high safety index, whereas an amino-derivative 7 showed the strongest antioxidant activity. Additionally, quantitative structure-activity relationship (QSAR) study demonstrated that mass, polarizability, topological charge, and van der Waals volume are essential properties governing the anti-MRSA activity. Taken together, cloxyquin was highlighted as a promising compound for further development as a novel anti-MRSA agent. QSAR findings would also benefit for further rational design of novel 8HQ-based compounds to combat the MRSA resistance.

Chemically Modified Derivatives of the Activator Compound Cloxyquin Exert Inhibitory Effect on TRESK (K2P18.1) Background Potassium Channel.

Cloxyquin has been reported as a specific activator of TRESK [TWIK-related spinal cord K+ channel (also known as K2P18.1)] background potassium channel. In this study, we have synthetized chemically modified analogs of cloxyquin and tested their effects on TRESK and other K2P channels. The currents of murine K2P channels, expressed heterologously in Xenopus oocytes, were measured by two-electrode voltage clamp, whereas the native background K+ conductance of mouse dorsal root ganglion (DRG) neurons was examined by the whole-cell patch-clamp method. Some of the analogs retained the activator character of the parent compound, but, more interestingly, other derivatives inhibited mouse TRESK current. The inhibitor analogs (A2764 and A2793) exerted state-dependent effects. The degree of inhibition by 100 µM A2764 (77.8% ± 3.5%, n = 6) was larger in the activated state of TRESK (i.e., after calcineurin-dependent stimulation) than in the resting state of the channel (42.8% ± 11.5% inhibition, n = 7). The selectivity of the inhibitor compounds was tested on several K2P channels. A2793 inhibited TWIK-related acid-sensitive K+ channel (TASK)-1 (100 µM, 53.4% ± 13, 5%, n = 5), while A2764 was more selective for TRESK, it only moderately influenced TREK-1 and TWIK-related alkaline pH-activated K+ channel. The effect of A2764 was also examined on the background K+ currents of DRG neurons. A subpopulation of DRG neurons, prepared from wild-type animals, expressed background K+ currents sensitive to A2764, whereas the inhibitor did not affect the currents in the DRG neurons of TRESK-deficient mice. Accordingly, A2764 may prove to be useful for the identification of TRESK current in native cells, and for the investigation of the role of the channel in nociception and migraine. SIGNIFICANCE STATEMENT: TRESK background potassium channel is a potential pharmacological target in migraine and neuropathic pain. In this study, we have identified a selective inhibitor of TRESK, A2764. This compound can inhibit TRESK in native cells, leading to cell depolarization and increased excitability. This new inhibitor may be of use to probe the role of TRESK channel in migraine and nociception.

Lysosome Membrane Permeabilization and Disruption of the Molecular Target of Rapamycin (mTOR)-Lysosome Interaction Are Associated with the Inhibition of Lung Cancer Cell Proliferation by a Chloroquinoline Analog.

Lysosomes degrade cellular proteins and organelles and regulate cell signaling by providing a surface for the formation of critical protein complexes, notably molecular target of rapamycin (mTOR) complex 1 (mTORC1). Striking differences in the lysosomes of cancer versus normal cells suggest that they could be targets for drug development. Although the lysomotropic drugs chloroquine (CQ) and hydroxychloroquine (HCQ) have been widely investigated, studies have focused on their ability to inhibit autophagy. We synthesized a novel compound, called EAD1, which is structurally related to CQ but is a 14-fold more potent inhibitor of cell proliferation. Here we find that EAD1 causes rapid relocation, membrane permeabilization (LMP), and deacidification of lysosomes, and it induces apoptosis and irreversibly blocks proliferation of human lung cancer H460, H520, H1299, HCC827, and H1703 cells. EAD1 causes dissociation of mTOR from lysosomes and increases mTOR's perinuclear versus cytoplasmic localization, changes previously shown to inactivate mTORC1. The effect on mTOR was not seen with HCQ, even at >10-fold greater concentrations. Phosphorylation of a downstream target of mTORC1, ribosomal protein S6, was inhibited by EAD1. Although EAD1 also inhibited autophagy, it retained full antiproliferative activity in autophagy-deficient H1650 lung cancer cells, which have a biallelic deletion of Atg7, and in H460 Atg7-knockout cells. As Atg7 is critical for the canonical autophagy pathway, it is likely that inhibition of autophagy is not how EAD1 inhibits cell proliferation. Further studies are needed to determine the relationship of LMP to mTORC1 disruption and their relative contributions to drug-induced cell death. These studies support the lysosome as an underexplored target for new drug development.

Evaluation of the in vitro and in vivo antileishmanial activity of a chloroquinolin derivative against Leishmania species capable of causing tegumentary and visceral leishmaniasis.

The treatment against leishmaniasis presents problems, since the currently used drugs are toxic and/or have high costs. In addition, parasite resistance has increased. As a consequence, in this study, a chloroquinolin derivative, namely 7-chloro-N,N-dimethylquinolin-4-amine or GF1059, was in vitro and in vivo tested against Leishmania parasites. Experiments were performed to evaluate in vitro antileishmanial activity and cytotoxicity, as well as the treatment of infected macrophages and the inhibition of infection using pre-treated parasites. This study also investigated the GF1059 mechanism of action in L. amazonensis. Results showed that the compound was highly effective against L. infantum and L. amazonensis, presenting a selectivity index of 154.6 and 86.4, respectively, against promastigotes and of 137.6 and 74.3, respectively, against amastigotes. GF1059 was also effective in the treatment of infected macrophages and inhibited the infection of these cells when parasites were pre-incubated with it. The molecule also induced changes in the parasites' mitochondrial membrane potential and cell integrity, and caused an increase in the reactive oxygen species production in L. amazonensis. Experiments performed in BALB/c mice, which had been previously infected with L. amazonensis promastigotes, and thus treated with GF1059, showed that these animals presented significant reductions in the parasite load when the infected tissue, spleen, liver, and draining lymph node were evaluated. GF1059-treated mice presented both lower parasitism and low levels of enzymatic markers, as compared to those receiving amphotericin B, which was used as control. In conclusion, data suggested that GF1059 can be considered a possible therapeutic target to be tested against leishmaniasis.

The cardioprotective compound cloxyquin uncouples mitochondria and induces autophagy.

Mitochondrial quality control mechanisms have been implicated in protection against cardiac ischemia-reperfusion (IR) injury. Previously, cloxyquin (5-chloroquinolin-8-ol) was identified via phenotypic screening as a cardioprotective compound. Herein, cloxyquin was identified as a mitochondrial uncoupler in both isolated heart mitochondria and adult cardiomyocytes. Additionally, cardiomyocytes isolated from transgenic mice expressing green fluorescent protein-tagged microtubule-associated protein light chain 3 showed increased autophagosome formation with cloxyquin treatment. The autophagy inhibitor chloroquine abolished cloxyquin-induced cardioprotection in both cellular and perfused heart (Langendorff) models of IR injury. Finally, in an in vivo murine left anterior descending coronary artery occlusion model of IR injury, cloxyquin significantly reduced infarct size from 31.4 ± 3.4% to 16.1 ± 2.2%. In conclusion, the cardioprotective compound cloxyquin simultaneously uncoupled mitochondria and induced autophagy. Importantly, autophagy appears to be required for cloxyquin-induced cardioprotection.

Characterisation of artemisinin-chloroquinoline hybrids for potential metabolic liabilities.

1. Chemotherapy remains the effective way of controlling malaria infections. Many of the treatments have been rendered ineffective as a result of drug resistance by plasmodia species as well as toxicity. Molecular hybridisation is one of the techniques used in the synthesis of new-generation antimalarial techniques. In this paper, we explore some potential metabolic challenges associated with this technique. 2. In vitro metabolic clearance and metabolite identification were performed in cryopreserved hepatocytes. Reaction phenotyping and inhibition studies were conducted in human liver microsomes and recombinant cytochrome P450s (CYPs) 3. Metabolism in hepatocytes was not extensive with less than 25% of the hybrids being metabolised by contributing CYP enzymes. The hybrids were, however, potent inhibitors of CYPs 2C9 2C19 and 3A4. 4. Our data indicated that artemisinin-chloroquinoline hybrid both gained and lost favourable properties from the individual pharmacophoric units from which they were built. This highlights the challenges associated with the molecular hybridisation technique and a need to optimise the chemistry in an effort to maintain good properties while addressing new liabilities that arise.

Toxin GhoT of the GhoT/GhoS toxin/antitoxin system damages the cell membrane to reduce adenosine triphosphate and to reduce growth under stress.

Toxin/antitoxin (TA) systems perhaps enable cells to reduce their metabolism to weather environmental challenges although there is little evidence to support this hypothesis. Escherichia coli GhoT/GhoS is a TA system in which toxin GhoT expression is reduced by cleavage of its messenger RNA (mRNA) by antitoxin GhoS, and TA system MqsR/MqsA controls GhoT/GhoS through differential mRNA decay. However, the physiological role of GhoT has not been determined. We show here through transmission electron microscopy, confocal microscopy and fluorescent stains that GhoT reduces metabolism by damaging the membrane and that toxin MqsR (a 5'-GCU-specific endoribonuclease) causes membrane damage in a GhoT-dependent manner. This membrane damage results in reduced cellular levels of ATP and the disruption of proton motive force (PMF). Normally, GhoT is localized to the pole and does not cause cell lysis under physiological conditions. Introduction of an F38R substitution results in loss of GhoT toxicity, ghost cell production and membrane damage while retaining the pole localization. Also, deletion of ghoST or ghoT results in significantly greater initial growth in the presence of antimicrobials. Collectively, these results demonstrate that GhoT reduces metabolism by reducing ATP and PMF and that this reduction in metabolism is important for growth with various antimicrobials.

Chloroxine inhibits pancreatic cancer progression through targeted antagonization of the PI3K/AKT/mTOR signaling pathway.

BACKGROUND: Patients with pancreatic cancer have a dismal prognosis due to tumor cell infiltration and metastasis. Many reports have documented that EMT and PI3K-AKT-mTOR axis control pancreatic cancer cell infiltration and metastasis. Chloroxine is an artificially synthesized antibacterial compound that demonstrated anti-pancreatic cancer effects in our previous drug-screening trial. We have explored the impact of chloroxine on pancreatic cancer growth, infiltration, migration, and apoptosis. METHODS: The proliferation of pancreatic cancer cell lines (PCCs) treated with chloroxine was assessed through real-time cell analysis (RTCA), colony formation assay, CCK-8 assay, as well as immunofluorescence. Chloroxine effects on the infiltrative and migratory capacities of PCCs were assessed via Transwell invasion and scratch experiments. To assess the contents of EMT- and apoptosis-associated proteins in tumor cells, we adopted Western immunoblotting as well as immunofluorescence assays, and flow cytometry to determine chloroxine effects on PCCs apoptosis. The in vivo chloroxine antineoplastic effects were explored in nude mice xenografts. RESULTS: Chloroxine repressed pancreatic cancer cell growth, migration, and infiltration in vitro, as well as in vivo, and stimulated apoptosis of the PCCs. Chloroxine appeared to inhibit PCC growth by Ki67 downregulation; this targeted and inhibited aberrant stimulation of the PI3K-AKT-mTOR signaling cascade, triggered apoptosis in PCC via mitochondria-dependent apoptosis, and modulated the EMT to inhibit PCC infiltration and migration. CONCLUSIONS: Chloroxine targeted and inhibited the PI3K-AKT-mTOR cascade to repress PCCs growth, migration, as well as invasion, and triggered cellular apoptosis. Therefore, chloroxine may constitute a potential antineoplastic drug for the treatment of pancreatic cancer.

Cloxyquin (5-chloroquinolin-8-ol) is an activator of the two-pore domain potassium channel TRESK.

TRESK is a two-pore domain potassium channel. Loss of function mutations have been linked to typical migraine with aura and due to TRESK's expression pattern and role in neuronal excitability it represents a promising therapeutic target. We developed a cell based assay using baculovirus transduced U20S cells to screen for activators of TRESK. Using a thallium flux system to measure TRESK channel activity we identified Cloxyquin as a novel activator. Cloxyquin was shown to have an EC50 of 3.8 µM in the thallium assay and displayed good selectivity against other potassium channels tested. Activity was confirmed using whole cell patch electrophysiology, with Cloxyquin causing a near two fold increase in outward current. The strategy presented here will be used to screen larger compound libraries with the aim of identifying novel chemical series which may be developed into new migraine prophylactics.

Cloxyquin activates hTRESK by allosteric modulation of the selectivity filter.

The TWIK-related spinal cord K+ channel (TRESK, K2P18.1) is a K2P channel contributing to the maintenance of membrane potentials in various cells. Recently, physiological TRESK function was identified as a key player in T-cell differentiation rendering the channel a new pharmacological target for treatment of autoimmune diseases. The channel activator cloxyquin represents a promising lead compound for the development of a new class of immunomodulators. Identification of cloxyquin binding site and characterization of the molecular activation mechanism can foster the future drug development. Here, we identify the cloxyquin binding site at the M2/M4 interface by mutational scan and analyze the molecular mechanism of action by protein modeling as well as in silico and in vitro electrophysiology using different permeating ion species (K+ / Rb+). In combination with kinetic analyses of channel inactivation, our results suggest that cloxyquin allosterically stabilizes the inner selectivity filter facilitating the conduction process subsequently activating hTRESK.

Antiplasmodial activity of novel keto-enamine chalcone-chloroquine based hybrid pharmacophores.

A series of novel keto-enamine chalcone-chloroquine based hybrids were synthesized following new methodology developed in our laboratory. The synthesized compounds were screened against chloroquine sensitive strain (3D7) of Plasmodium falciparum in an in vitro model. Some of the compounds were showing comparable antimalarial activity at par with chloroquine. Compounds with significant in vitro antimalarial activity were then evaluated for their in vivo efficacy in Swiss mice against Plasmodium yoelii (chloroquine resistant N-67 strain), wherein compounds 25 and 27 each showed an in vivo suppression of 99.9% parasitaemia on day 4. Biochemical studies reveal that inhibition of hemozoin formation is the primary mechanism of action of these analogues.

Drug screening to identify compounds to act as co-therapies for the treatment of Burkholderia species.

Burkholderia pseudomallei is a soil-dwelling organism present throughout the tropics. It is the causative agent of melioidosis, a disease that is believed to kill 89,000 people per year. It is naturally resistant to many antibiotics, requiring at least two weeks of intravenous treatment with ceftazidime, imipenem or meropenem followed by 6 months of orally delivered co-trimoxazole. This places a large treatment burden on the predominantly middle-income nations where the majority of disease occurs. We have established a high-throughput assay for compounds that could be used as a co-therapy to potentiate the effect of ceftazidime, using the related non-pathogenic bacterium Burkholderia thailandensis as a surrogate. Optimization of the assay gave a Z' factor of 0.68. We screened a library of 61,250 compounds and identified 29 compounds with a pIC50 (-log10(IC50)) greater than five. Detailed investigation allowed us to down select to six "best in class" compounds, which included the licensed drug chloroxine. Co-treatment of B. thailandensis with ceftazidime and chloroxine reduced culturable cell numbers by two orders of magnitude over 48 hours, compared to treatment with ceftazidime alone. Hit expansion around chloroxine was performed using commercially available compounds. Minor modifications to the structure abolished activity, suggesting that chloroxine likely acts against a specific target. Finally, an initial study demonstrates the utility of chloroxine to act as a co-therapy to potentiate the effect of ceftazidime against B. pseudomallei. This approach successfully identified potential co-therapies for a recalcitrant Gram-negative bacterial species. Our assay could be used more widely to aid in chemotherapy to treat infections caused by these bacteria.

Repurposing clinically approved drugs for COVID-19 treatment targeting SARS-CoV-2 papain-like protease.

COVID-19 is a disease caused by SARS-CoV-2, which has led to more than 4 million deaths worldwide. As a result, there is a worldwide effort to develop specific drugs for targeting COVID-19. Papain-like protease (PLpro) is an attractive drug target because it has multiple essential functions involved in processing viral proteins, including viral genome replication and removal of post-translational ubiquitination modifications. Here, we established two assays for screening PLpro inhibitors according to protease and anti-ISGylation activities, respectively. Application of the two screening techniques to the library of clinically approved drugs led to the discovery of tanshinone IIA sulfonate sodium and chloroxine with their IC50 values of lower than 10 µM. These two compounds were found to directly interact with PLpro and their molecular mechanisms of binding were illustrated by docking and molecular dynamics simulations. The results highlight the usefulness of the two developed screening techniques for locating PLpro inhibitors.

Chloroxine overrides DNA damage tolerance to restore platinum sensitivity in high-grade serous ovarian cancer.

High-grade serous cancer (HGSC) accounts for ~67% of all ovarian cancer deaths. Although initially sensitive to platinum chemotherapy, resistance is inevitable and there is an unmet clinical need for novel therapies that can circumvent this event. We performed a drug screen with 1177 FDA-approved drugs and identified the hydroxyquinoline drug, chloroxine. In extensive validation experiments, chloroxine restored sensitivity to both cisplatin and carboplatin, demonstrating broad synergy in our range of experimental models of platinum-resistant HGSC. Synergy was independent of chloroxine's predicted ionophore activity and did not relate to platinum uptake as measured by atomic absorption spectroscopy. Further mechanistic investigation revealed that chloroxine overrides DNA damage tolerance in platinum-resistant HGSC. Co-treatment with carboplatin and chloroxine (but not either drug alone) caused an increase in γH2AX expression, followed by a reduction in platinum-induced RAD51 foci. Moreover, this unrepaired DNA damage was associated with p53 stabilisation, cell cycle re-entry and triggering of caspase 3/7-mediated cell death. Finally, in our platinum-resistant, intraperitoneal in vivo model, treatment with carboplatin alone resulted in a transient tumour response followed by tumour regrowth. In contrast, treatment with chloroxine and carboplatin combined, was able to maintain tumour volume at baseline for over 4 months. In conclusion, our novel results show that chloroxine facilitates platinum-induced DNA damage to restore platinum sensitivity in HGSC. Since chloroxine is already licensed, this exciting combination therapy could now be rapidly translated for patient benefit.

RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca2+ assays.

Elevated cytoplasmic [Ca2+] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca2+ leak from sarcoplasmic reticulum stores via dysregulated ryanodine receptor (RyR) channels. Consequently, RyR is recognized as a high-value target for drug discovery to treat such pathologies. Using a FRET-based high-throughput screening assay that we previously reported, we identified small-molecule compounds that modulate the skeletal muscle channel isoform (RyR1) interaction with calmodulin and FK506 binding protein 12.6. Two such compounds, chloroxine and myricetin, increase FRET and inhibit [3H]ryanodine binding to RyR1 at nanomolar Ca2+. Both compounds also decrease RyR1 Ca2+ leak in human skinned skeletal muscle fibers. Furthermore, we identified compound concentrations that reduced leak by > 50% but only slightly affected Ca2+ release in excitation-contraction coupling, which is essential for normal muscle contraction. This report demonstrates a pipeline that effectively filters small-molecule RyR1 modulators towards clinical relevance.

Mechanical factors tune the sensitivity of mdx muscle to eccentric strength loss and its protection by antioxidant and calcium modulators.

BACKGROUND: Dystrophin deficiency sensitizes skeletal muscle of mice to eccentric contraction (ECC)-induced strength loss. ECC protocols distinguish dystrophin-deficient from healthy, wild type muscle, and test the efficacy of therapeutics for Duchenne muscular dystrophy (DMD). However, given the large lab-to-lab variability in ECC-induced strength loss of dystrophin-deficient mouse skeletal muscle (10-95%), mechanical factors of the contraction likely impact the degree of loss. Therefore, the purpose of this study was to evaluate the extent to which mechanical variables impact sensitivity of dystrophin-deficient mouse skeletal muscle to ECC. METHODS: We completed ex vivo and in vivo muscle preparations of the dystrophin-deficient mdx mouse and designed ECC protocols within physiological ranges of contractile parameters (length change, velocity, contraction duration, and stimulation frequencies). To determine whether these contractile parameters affected known factors associated with ECC-induced strength loss, we measured sarcolemmal damage after ECC as well as strength loss in the presence of the antioxidant N-acetylcysteine (NAC) and small molecule calcium modulators that increase SERCA activity (DS-11966966 and CDN1163) or lower calcium leak from the ryanodine receptor (Chloroxine and Myricetin). RESULTS: The magnitude of length change, work, and stimulation duration ex vivo and in vivo of an ECC were the most important determinants of strength loss in mdx muscle. Passive lengthening and submaximal stimulations did not induce strength loss. We further showed that sarcolemmal permeability was associated with muscle length change, but it only accounted for a minimal fraction (21%) of the total strength loss (70%). The magnitude of length change also significantly influenced the degree to which NAC and small molecule calcium modulators protected against ECC-induced strength loss. CONCLUSIONS: These results indicate that ECC-induced strength loss of mdx skeletal muscle is dependent on the mechanical properties of the contraction and that mdx muscle is insensitive to ECC at submaximal stimulation frequencies. Rigorous design of ECC protocols is critical for effective use of strength loss as a readout in evaluating potential therapeutics for muscular dystrophy.

Reduction of dopamine synaptic activity: degradation of 50-kHz ultrasonic vocalization in rats.

Vocal deficits are prevalent and debilitating in Parkinson's disease. These deficits may be related to the initial pathology of the nigrostriatal dopamine neurons and resulting dopamine depletion, which contributes to dysfunction of fine motor control in multiple functions. Although vocalization in animals and humans may differ in many respects, we evaluated complex (50-kHz) ultrasonic mate calls in 2 rat models of Parkinson's disease, including unilateral infusions of 6-hydroxydopamine to the medial forebrain bundle and peripheral administration of a nonakinesia dose of the dopamine antagonist haloperidol. We examined the effects of these treatments on multiple aspects of the acoustic signal. The number of trill-like (frequency modulated) 50-kHz calls was significantly reduced, and appeared to be replaced by simpler (flat) calls. The bandwidth and maximum intensity of simple and frequency-modulated calls were significantly decreased, but call duration was not. Our findings suggest that the nigrostriatal dopamine pathway is involved to some extent in fine sensorimotor function that includes USV production and complexity.

Development of p53-targeting drugs that increase radioresistance in normal tissues.

Radiation damage to normal tissues is a serious concern in radiation therapy. Advances in radiotherapeutic technology have improved the dose distribution of the target volumes and risk organs, but damage to risk organs that are located within the irradiation field still limits the allowable prescription dose. To overcome this dose-limiting toxicity, and to further improve the efficacy of radiotherapy, the development of drugs that protect normal tissues but not cancer tissues from the effects of radiation are expected to be developed based on molecular target-based drugs. p53 is a well-known transcription factor that is closely associated with radiation-induced cell death. In radiation-injured tissues, p53 induces apoptosis in hematopoietic lineages, whereas it plays a radioprotective role in the gastrointestinal epithelium. These facts suggest that p53 inhibitor would be effective for radioprotection of the hematopoietic system, and that a drug that upregulates the radioprotective functions of p53 would enhance the radioresistance of gastrointestinal tissues. In this review, we summarize recent progress regarding the prevention of radiation injury by regulating p53 and provide new strategic insights into the development of radioprotectors in radiotherapy. J. Med. Invest. 66 : 219-223, August, 2019.

Cloxiquine, a traditional antituberculosis agent, suppresses the growth and metastasis of melanoma cells through activation of PPARγ.

Melanoma is one of the most aggressive skin cancers and 5-year survival rate is only 4.6% for metastatic melanoma patients. Current therapies, especially those involving clinical chemotherapy drugs, have achieved remarkable advances. However, their side effects, such as bone marrow suppression, limit the effectiveness of available pharmacological therapies. Therefore, exploring new antimelanoma drugs with less toxicity is critical for the treatment of melanoma. In the present study, we aimed to identify the antimelanoma drugs with ability to repress the proliferation of melanoma cells by using a high-content screening of FDA-approved drug libraries. We found that cloxiquine (CLQ), a traditional antituberculosic drug, exhibited strong inhibitory effects on the growth and metastasis of melanoma cells both in vivo and in vitro. In contrast, CLQ at the tested doses did not show any apparent toxicity in normal melanocytes and in the liver. At the metabolic level, treatment with CLQ decreased glycolysis, thus potentially inhibiting the "Warburg effect" in B16F10 cells. More importantly, combination of CLQ and 2-deoxyglucose (2-DG), a well-known glycolysis inhibitor, did not show a synergistic effect on the tumor growth and metastasis, indicating that inhibition of glycolysis is potentially involved in mediating CLQ's antimelanoma function. Bioinformatics analyses revealed that peroxisome proliferator-activated receptor-gamma (PPARγ) served as a potential CLQ target. Mechanistically, CLQ stimulated the transcription and nuclear contents of PPARγ. Furthermore, the specific PPARγ inhibitor GW9662 or PPARγ shRNA partially abolished the effects of CLQ. Collectively, our findings demonstrate that CLQ has a great potential in the treatment of melanoma through activation of PPARγ.