Activation of Transcription Factor Nrf2 in HeLa Cells under the Action of Nitrosyl Iron Complex with N-Ethylthiourea.

We studied the effect of an NO donor, nitrosyl iron complex with N-ethylthiourea, on Nrf2-dependent antioxidant system activation of tumor cells in vitro. The complex increased intracellular accumulation of Nrf2 transcription factor and induced its nuclear translocation. It was shown that both heme oxygenase-1 gene and protein expression increased significantly under the influence of the complex. Nrf2 activation was accompanied by a decrease in the intracellular accumulation of proinflammatory transcription factor NF-κB p65 subunit and expression of its target genes. The cytotoxic effect of N-ethylthiourea leads to induction of Nrf2/HO-1 antioxidant response and suppression of NF-κB-dependent processes in tumor cells.

In vivo effects of a selected thiourea derivative 1-(2-chlorobenzoyl)-3-(2,3-dichlorophenyl) against nociception, inflammation and gastric ulcerogenicity: Biochemical, histopathological and in silico approaches.

The current study was designed to investigate the potential of a synthetic therapeutic agent for better management of pain and inflammation, exhibiting minimal to non-existent ulcerogenic effects. The effect of 1-(2-chlorobenzoyl)-3-(2,3-dichlorophenyl) thiourea was assessed through model systems of nociception and anti-inflammatory activities in mice. In addition, the ulcerogenic potential was evaluated in rats using the NSAID-induced pyloric ligation model, followed by histopathological and biochemical analysis. The test was conducted on eight groups of albino rats, comprising of group I (normal saline), groups II and III (aspirin® at doses of 100 mg/kg and 150 mg/kg, respectively), groups IV and V (indomethacin at doses of 100 mg/kg and 150 mg/kg, respectively), and groups VI, VII, and VIII (lead-compound at 15 mg/kg, 30 mg/kg and 45 mg/kg doses, respectively). Furthermore, molecular docking analyses were performed to predict potential molecular target site interactions. The results showed that the lead-compound, administered at doses of 15, 30, and 45 mg/kg, yielded significant reductions in chemically and thermally induced nociceptive pain, aligning with the levels observed for aspirin® and tramadol. The compound also effectively suppressed inflammatory response in the carrageenan-induced paw edema model. As for the ulcerogenic effects, the compound groups displayed no considerable alterations compared to the aspirin® and indomethacin groups, which displayed substantial increases in ulcer scores, total acidity, free acidity, and gastric juice volume, and a decrease in gastric juice pH. In conclusion, these findings suggest that our test compound exhibits potent antinociceptive, anti-inflammatory properties and is devoid of ulcerogenic effects.

Inhibition of the Sodium-Calcium Exchanger Reverse Mode Activity Reduces Alcohol Consumption in Rats.

Excessive and uncontrolled consumption of alcohol can cause alcohol use disorder (AUD), but its pharmacological mechanisms are not fully understood. Inhibiting the reverse mode activity of the sodium-calcium exchanger (NCX) can reduce the risk of alcohol withdrawal seizures, suggesting that NCX could play a role in controlling alcohol consumption. Here, we investigated how two potent inhibitors of NCX reverse mode activity, SN-6 (NCX1) and KB-R7943 (NCX3), affect voluntary alcohol consumption in adult male and female rats using the intermittent alcohol access two-bottle choice paradigm. Initially, animals were trained to drink 7.5% ethanol and water for four weeks before administering SN-6 and KB-R7934. Afterward, their alcohol intake, preference, and water intake were recorded 2 and 24 h after exposure to water and 7.5% ethanol. SN-6 significantly reduced alcohol consumption by 48% in male and 36% in female rats without affecting their water intake. Additionally, SN-6 significantly reduced alcohol preference in females by 27%. However, KB-R7943 reduced alcohol consumption by 42% in female rats and did not affect alcohol preference or water intake. These findings suggest that alcohol exposure increased NCX reverse activity, and targeting NCX1 could be an effective strategy for reducing alcohol consumption in subjects susceptible to withdrawal seizures.

Salubrinal promotes phospho-eIF2α-dependent activation of UPR leading to autophagy-mediated attenuation of iron-induced insulin resistance.

Identification of new mechanisms mediating insulin sensitivity is important to allow validation of corresponding therapeutic targets. In this study, we first used a cellular model of skeletal muscle cell iron overload and found that endoplasmic reticulum (ER) stress and insulin resistance occurred after iron treatment. Insulin sensitivity was assessed using cells engineered to express an Akt biosensor, based on nuclear FoxO localization, as well as western blotting for insulin signaling proteins. Use of salubrinal to elevate eIF2α phosphorylation and promote the unfolded protein response (UPR) attenuated iron-induced insulin resistance. Salubrinal induced autophagy flux and its beneficial effects on insulin sensitivity were not observed in autophagy-deficient cells generated by overexpressing a dominant-negative ATG5 mutant or via knockout of ATG7. This indicated the beneficial effect of salubrinal-induced UPR activation was autophagy-dependent. We translated these observations to an animal model of systemic iron overload-induced skeletal muscle insulin resistance where administration of salubrinal as pretreatment promoted eIF2α phosphorylation, enhanced autophagic flux in skeletal muscle and improved insulin responsiveness. Together, our results show that salubrinal elicited an eIF2α-autophagy axis leading to improved skeletal muscle insulin sensitivity both in vitro and in mice.

Structural basis of ligand recognition and design of antihistamines targeting histamine H4 receptor.

The histamine H4 receptor (H4R) plays key role in immune cell function and is a highly valued target for treating allergic and inflammatory diseases. However, structural information of H4R remains elusive. Here, we report four cryo-EM structures of H4R/Gi complexes, with either histamine or synthetic agonists clobenpropit, VUF6884 and clozapine bound. Combined with mutagenesis, ligand binding and functional assays, the structural data reveal a distinct ligand binding mode where D943.32 and a π-π network determine the orientation of the positively charged group of ligands, while E1825.46, located at the opposite end of the ligand binding pocket, plays a key role in regulating receptor activity. The structural insight into H4R ligand binding allows us to identify mutants at E1825.46 for which the agonist clobenpropit acts as an inverse agonist and to correctly predict inverse agonism of a closely related analog with nanomolar potency. Together with the findings regarding receptor activation and Gi engagement, we establish a framework for understanding H4R signaling and provide a rational basis for designing novel antihistamines targeting H4R.

Novel insight on IRE1 in the regulation of chondrocyte dedifferentiation through ER stress independent pathway.

Inositol-requiring enzyme-1 (IRE1) is the master regulator of the unfolded protein response pathway, associated with the endoplasmic reticulum (ER) in sensing and regulating cell stress. The activity of IRE1 is highly explored and well-characterized in cancer and other cells. However, the IRE1 molecular mechanism in chondrocytes is poorly understood. The present study explored the effect of IRE1 on chondrocytes regarding its chondrogenic gene expression and its correlation with different cellular pathways and cell behavior. Chondrocytes transfected with the cDNA of IRE1 reduced the expression of type II collagen, disrupting chondrocyte differentiation as confirmed by western blotting and immunofluorescence. Upon siRNA treatment, the influence of IRE1 on chondrocyte differentiation is restored by reviving the normal expression of type II collagen. Different molecular pathways were explored to investigate the role of IRE1 in causing chondrocyte dedifferentiation. However, we found no significant correlation, as IRE1 induces dedifferentiation through independent pathways. In response to various endoplasmic reticulum (ER) agonists (2-deoxy-D-glucose), and ER stress antagonists (tauroursodeoxycholic acid and salubrinal), IRE1 overexpression did not affect GRP78/94, as implicated in the pathogenesis of ER stress. Moreover, when IRE1 overexpression was correlated with the inflammation pathway, nuclear factor-kappa B (NFκB), IRE1 substantially increased the expression of p50 while decreasing the expression of nuclear factor kappa light polypeptide alpha (IκBα). These results suggest that IRE1 induces dedifferentiation in chondrocytes by modulating inflammatory pathways that cause dedifferentiation by disrupting type II collagen expression.

PERK modulation, with GSK2606414, Sephin1 or salubrinal, failed to produce therapeutic benefits in the SOD1G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) has been linked to overactivity of the protein kinase RNA-like ER kinase (PERK) branch of the unfolded protein response (UPR) pathway, both in ALS patients and mouse models. However, attempts to pharmacologically modulate PERK for therapeutic benefit have yielded inconsistent and often conflicting results. This study sought to address these discrepancies by comprehensively evaluating three commonly used, CNS-penetrant, PERK modulators (GSK2606414, salubrinal, and Sephin1) in the same experimental models, with the goal of assessing the viability of targeting the PERK pathway as a therapeutic strategy for ALS. To achieve this goal, a tunicamycin-challenge assay was developed using wild-type mice to monitor changes in liver UPR gene expression in response to PERK pathway modulation. Subsequently, multiple dosing regimens of each PERK modulator were tested in standardized, well-powered, gender-matched, and litter-matched survival efficacy studies using the SOD1G93A mouse model of ALS. The alpha-2-adrenergic receptor agonist clonidine was also tested to elucidate the results obtained from the Sephin1, and of the previously reported guanabenz studies, by comparing the effects of presence or absence of α-2 agonism. The results revealed that targeting PERK may not be an ideal approach for ALS treatment. Inhibiting PERK with GSK2606414 or activating it with salubrinal did not confer therapeutic benefits. While Sephin1 showed some promising therapeutic effects, it appears that these outcomes were mediated through PERK-independent mechanisms. Clonidine also produced some favorable therapeutic effects, which were unexpected and not linked to the UPR. In conclusion, this study highlights the challenges of pharmacologically targeting PERK for therapeutic purposes in the SOD1G93A mouse model and suggests that exploring other targets within, and outside, the UPR may be more promising avenues for ALS treatment.

Lysosomal BK channels facilitate silica-induced inflammation in macrophages.

BACKGROUND: Lysosomal ion channels are proposed therapeutic targets for a number of diseases, including those driven by NLRP3 inflammasome-mediated inflammation. Here, the specific role of the lysosomal big conductance Ca2+-activated K+ (BK) channel was evaluated in a silica model of inflammation in murine macrophages. A specific-inhibitor of BK channel function, paxilline (PAX), and activators NS11021 and NS1619 were utilized to evaluate the role of lysosomal BK channel activity in silica-induced lysosomal membrane permeabilization (LMP) and NLRP3 inflammasome activation resulting in IL-1ß release. METHODS: Murine macrophages were exposed in vitro to crystalline silica following pretreatment with BK channel inhibitors or activators and LMP, cell death, and IL-1ß release were assessed. In addition, the effect of PAX treatment on silica-induced cytosolic K+ decrease was measured. Finally, the effects of BK channel modifiers on lysosomal pH, proteolytic activity, and cholesterol transport were also evaluated. RESULTS: PAX pretreatment significantly attenuated silica-induced cell death and IL-1ß release. PAX caused an increase in lysosomal pH and decrease in lysosomal proteolytic activity. PAX also caused a significant accumulation of lysosomal cholesterol. BK channel activators NS11021 and NS1619 increased silica-induced cell death and IL-1ß release. BK channel activation also caused a decrease in lysosomal pH and increase in lysosomal proteolytic function as well as a decrease in cholesterol accumulation. CONCLUSION: Taken together, these results demonstrate that inhibiting lysosomal BK channel activity with PAX effectively reduced silica-induced cell death and IL-1ß release. Blocking cytosolic K+ entry into the lysosome prevented LMP through the decrease of lysosomal acidification and proteolytic function and increase in lysosomal cholesterol.

Rational design and optimization of acylthioureas as novel potent influenza virus non-nucleoside polymerase inhibitors.

Evidence suggests that rapidly evolving virus subvariants risk rendering current vaccines and anti-influenza drugs ineffective. Hence, exploring novel scaffolds or new targets of anti-influenza drugs is of great urgency. Herein, we report the discovery of a series of acylthiourea derivatives produced via a scaffold-hopping strategy as potent antiviral agents against influenza A and B subtypes. The most effective compound 10m displayed subnanomolar activity against H1N1 proliferation (EC50 = 0.8 nM) and exhibited inhibitory activity toward other influenza strains, including influenza B virus and H1N1 variant (H1N1, H274Y). Additionally, druggability evaluation revealed that 10m exhibited favorable pharmacokinetic properties and was metabolically stable in liver microsome preparations from three different species as well as in human plasma. In vitro and in vivo toxicity studies confirmed that 10m demonstrated a high safety profile. Furthermore, 10m exhibited satisfactory antiviral activity in a lethal influenza virus mouse model. Moreover, mechanistic studies indicated that these acylthiourea derivatives inhibited influenza virus proliferation by targeting influenza virus RNA-dependent RNA polymerase. Thus, 10m is a potential lead compound for the further exploration of treatment options for influenza.

Inhibition of mycobacteria proliferation in macrophages by low cisplatin concentration through phosphorylated p53-related apoptosis pathway.

BACKGROUND: Drug resistance is a prominent problem in the treatment of tuberculosis, so it is urgent to develop new anti- tuberculosis drugs. Here, we investigated the effects and mechanisms of cisplatin (DDP) on intracellular Mycobacterium smegmatis to tap the therapeutic potential of DDP in mycobacterial infection. RESULTS: Macrophages infected with Mycobacterium smegmatis were treated with DDP alone or combined with isoniazid or rifampicin. The results showed that the bacterial count in macrophages decreased significantly after DDP (≤ 6 µg/mL) treatment. When isoniazid or rifampicin was combined with DDP, the number of intracellular mycobacteria was also significantly lower than that of isoniazid or rifampicin alone. Apoptosis of infected cells increased after 24 h of DDP treatment, as shown by flow cytometry and transmission electron microscopy detection. Transcriptome sequencing showed that there were 1161 upregulated and 645 downregulated differentially expressed genes (DEGs) between the control group and DDP treatment group. A Trp53-centered protein interaction network was found based on the top 100 significant DEGs through STRING and Cytoscape software. The expression of phosphorylated p53, Bax, JAK, p38 MAPK and PI3K increased after DDP treatment, as shown by Western blot analysis. Inhibitors of JAK, PI3K or p38 MAPK inhibited the increase in cell apoptosis and the reduction in the intracellular bacterial count induced by DDP. The p53 promoter Kevetrin hydrochloride scavenges intracellular mycobacteria. If combined with DDP, Kevetrin hydrochloride could increase the effect of DDP on the elimination of intracellular mycobacteria. In conclusion, DDP at low concentrations could activate the JAK, p38 MAPK and PI3K pathways in infected macrophages, promote the phosphorylation of p53 protein, and increase the ratio of Bax to Bcl-2, leading to cell apoptosis, thus eliminating intracellular bacteria and reducing the spread of mycobacteria. CONCLUSION: DDP may be a new host-directed therapy for tuberculosis treatment, as well as the p53 promoter Kevetrin hydrochloride.

New thiourea and benzamide derivatives of 2-aminothiazole as multi-target agents against Alzheimer's disease: Design, synthesis, and biological evaluation.

In this study, two series of compounds were designed and synthesized, bearing thiourea and benzamide derivatives at position 2 of 4-subtituted-2-aminothiazole, respectively. Then, the inhibition potency of all final compounds for cholinesterase enzymes were evaluated. Among the thiourea derivatives, 3c (IC50 = 0.33 µM) was identified as the most potent and selective butyrylcholinesterase inhibitor. Additionally, benzamide derivative 10e (AChE IC50 = 1.47 and BChE IC50 = 11.40 µM) was found as a dual cholinesterase inhibitor. The type of inhibition for both compounds was determined by kinetic studies and the results showed that the compounds were mixed type inhibitors. Moreover, all title compounds were investigated in terms of their antioxidant (DPHH, ORAC) and metal chelator activities. In addition, the neuroprotective effects of selected compounds (3c, 3e, 6c, 6e and 10e) against H2O2-induced damage in the PC12 cell line were tested. The experimental findings demonstrated that thiourea-derived 6e (40.4 %) and benzamide-derived 10e (37.8 %) have a neuroprotective effect of about half as ferulic acid at 10 µM. Subsequently, the cytotoxicity of selected compounds was examined by the MTT assay, and the compounds were found not to have cytotoxic effect on the PC12 cell line in 24 h. Additionally, compounds 6e and 10e were also found to be more effective in inhibiting the release of IL-1ß, IL-6, TNF-α and NO compared to other selected compounds in this study.

Derivatization-free determination of carbonyl compounds using bifunctional chemiluminescence coreactant thiourea dioxide.

Uniquely, thiourea dioxide not only can reduce carbonyl compounds but also generate an oxidant to trigger luminol chemiluminescence. Herein, derivatization-free carbonyl compound detection using bifunctional chemiluminescence coreactant thiourea dioxide has been developed for the first time with the second most crucial flavor benzaldehyde as a representative.

Effect of solvents and glutathione on the decomposition of the nitrosyl iron complex with N-ethylthiourea ligands: An experimental and theoretical study.

Dinitrosyl iron complexes (DNICs) are a depot and potential source of free NO in organisms. Their synthetic analog, N-ethylthiourea DNIC [Fe(SC(NH2)(NHC2H5))2(NO)2]+Cl-∙[Fe(SC(NH2)(NHC2H5))Cl(NO)2]0 (complex 1), as cardioprotective and cytostatic agent is a promising prodrug for the treatment of socially relevant diseases. In this work, transformation mechanism of complex 1 has been studied in anaerobic aqueous solution (pH = 7.0), DMSO, and ethanol. It was shown that the solvent has a significant effect on the decomposition of complex. According to EPR-spectroscopy, only cationic part of complex is found upon its dissolution in water; only neutral part is retained in DMSO, and both fragments are present in ethanol. Effective generation of NO occurs in an aqueous solution. The structures of the decomposition products were proposed for all solvents, their UV-spectra and rate constants were calculated. From the experimental and theoretical data obtained, it follows that complex 1 is most stable in DMSO. Solutions of complex in a DMSO-water mixture can be used to improve its bioavailability in further in vitro and in vivo studies. Also, we have analyzed its interaction with glutathione (GSH), which can participate in the metabolism of this compound. This study shows that complex 1 reacts with GSH to form a new binuclear DNIC with two GS--ligands. It was found that the resulting complex is a more prolonged NO-donor than the initial one: k = 6.1∙10-3·s-1 in buffer, k = 6.4∙10-5 s-1 with GSH. This reaction may prevent S-glutathionylation of the essential enzyme systems and is important for metabolism of complex, associated with its antitumor activity.

Salubrinal induces fetal hemoglobin expression via the stress-signaling pathway in human sickle erythroid progenitors and sickle cell disease mice.

Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the HBB gene leading to hemoglobin S production and polymerization under hypoxia conditions leading to vaso-occlusion, chronic hemolysis, and progressive organ damage. This disease affects ~100,000 people in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as hydroxyurea, the only Food and Drug Administration-approved drug for this purpose. Therefore, the goal of our study was to determine whether salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression through the stress-signaling pathway by activation of p-eIF2α and ATF4 trans-activation in the γ-globin gene promoter. Sickle erythroid progenitors treated with 24µM SAL increased F-cells levels 1.4-fold (p = 0.021) and produced an 80% decrease in reactive oxygen species. Western blot analysis showed SAL enhanced HbF protein by 1.6-fold (p = 0.0441), along with dose-dependent increases of p-eIF2α and ATF4 levels. Subsequent treatment of SCD mice by a single intraperitoneal injection of SAL (5mg/kg) produced peak plasma concentrations at 6 hours. Chronic treatments of SCD mice with SAL mediated a 2.3-fold increase in F-cells (p = 0.0013) and decreased sickle erythrocytes supporting in vivo HbF induction.

Salubrinal-mediated activation of eIF2α signaling improves oxidative stress-induced BMSCs senescence and senile osteoporosis.

Bone cells of various lineages become senescent in bone microenvironment. Senotherapies that clear the senescent bone cells improve bone microarchitecture of aged bones. However, the mechanisms underlie for the formation and maintenance of senescent bone cells are largely unknown. Here, we focus on the relationship between endoplasmic reticulum stress (ER stress)-activated unfolded protein response (UPR) signaling and cellular senescence of bone marrow mesenchymal stem cells (BMSCs). The PKR-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2 α(eIF2α) signaling branch was specifically activated and tightly regulated in senescent BMSCs induced by hydrogen peroxide (H2O2). However, blocking PERK-eIF2α signaling with AMG'44 could not reverse the cellular senescence phenotype of senescent BMSCs. Treated the senescent cells with salubrinal, an inhibitor for dephosphorylation of eIF2α, decreased SA-ß-Gal positive cells and the expression of markers for cellular senescence. Moreover, salubrinal enhanced the apoptosis of senescent BMSCs and upregulated expression of Chop and BIM. Furthermore, salubrinal treatment significantly improved the osteogenesis capacity of senescent BMSCs as reflected by the increase of Alp, Runx2 and Osteocalcin, the formation of Alp-positive staining cells and matrix mineralization. Salubrinal administration results in significant recovery in the bone microarchitecture of senile SAMP6 mice. Taken together, our data reveal an undefined role of PERK-eIF2α signaling in the maintenance of cellular senescent phenotype in BMSCs. The activation of eIF2α signaling with salubrinal is helpful for the clearance of senescent BMSCs and the improvement of bone integrity of aged mice.

Cell density-dependent antibiotic tolerance to inhibition of the elongation machinery requires fully functional PBP1B.

The peptidoglycan (PG) cell wall provides shape and structure to most bacteria. There are two systems to build PG in rod shaped organisms: the elongasome and divisome, which are made up of many proteins including the essential MreB and PBP2, or FtsZ and PBP3, respectively. The elongasome is responsible for PG insertion during cell elongation, while the divisome is responsible for septal PG insertion during division. We found that the main elongasome proteins, MreB and PBP2, can be inhibited without affecting growth rate in a quorum sensing-independent density-dependent manner. Before cells reach a particular cell density, inhibition of the elongasome results in different physiological responses, including intracellular vesicle formation and an increase in cell size. This inhibition of MreB or PBP2 can be compensated for by the presence of the class A penicillin binding protein, PBP1B. Furthermore, we found this density-dependent growth resistance to be specific for elongasome inhibition and was consistent across multiple Gram-negative rods, providing new areas of research into antibiotic treatment.

Involvement of Histamine H3 Receptor Agonism in Premature Ejaculation Found by Studies in Rats.

Several of the drugs currently available for the treatment of premature ejaculation (PE) (e.g., local anesthetics or antidepressants) are associated with numerous safety concerns and exhibit weak efficacy. To date, no therapeutics for PE have been approved in the United States, highlighting the need to develop novel agents with sufficient efficacy and fewer side effects. In this study, we focused on the histamine H3 receptor (H3R) as a potential target for the treatment of PE and evaluated the effects of imetit (an H3R/H4R agonist), ciproxifan (an H3R antagonist), and JNJ-7777120 (an H4R antagonist) in vivo. Our in vivo electrophysiological experiments revealed that imetit reduced mechanical stimuli-evoked neuronal firing in anesthetized rats. This effect was inhibited by ciproxifan but not by JNJ-7777120. Subsequently, we evaluated the effect of imetit using a copulatory behavior test to assess ejaculation latency (EL) in rats. Imetit prolonged EL, although this effect was inhibited by ciproxifan. These findings indicate that H3R stimulation suppresses mechanical stimuli-evoked neuronal firing in the spinal-penile neurotransmission system, thereby resulting in prolonged EL. To our knowledge, this is the first report to describe the relationship between H3R and PE. Thus, H3R agonists may represent a novel treatment option for PE.

Suppressing Effect of Na+/Ca2+ Exchanger (NCX) Inhibitors on the Growth of Melanoma Cells.

The role of calcium ion (Ca2+) signaling in tumorigenicity has received increasing attention in melanoma research. Previous Ca2+ signaling studies focused on Ca2+ entry routes, but rarely explored the role of Ca2+ extrusion. Functioning of the Na+/Ca2+ exchanger (NCX) on the plasma membrane is the major way of Ca2+ extrusion, but very few associations between NCX and melanoma have been reported. Here, we explored whether pharmacological modulation of the NCX could suppress melanoma and promise new therapeutic strategies. Methods included cell viability assay, Ca2+ imaging, immunoblotting, and cell death analysis. The NCX inhibitors SN-6 and YM-244769 were used to selectively block reverse operation of the NCX. Bepridil, KB-R7943, and CB-DMB blocked either reverse or forward NCX operation. We found that blocking the reverse NCX with SN-6 or YM-244769 (5-100 µM) did not affect melanoma cells or increase cytosolic Ca2+. Bepridil, KB-R7943, and CB-DMB all significantly suppressed melanoma cells with IC50 values of 3-20 µM. Bepridil and KB-R7943 elevated intracellular Ca2+ level of melanoma. Bepridil-induced melanoma cell death came from cell cycle arrest and enhanced apoptosis, which were all attenuated by the Ca2+ chelator BAPTA-AM. As compared with melanoma, normal melanocytes had lower NCX1 expression and were less sensitive to the cytotoxicity of bepridil. In conclusion, blockade of the forward but not the reverse NCX leads to Ca2+-related cell death in melanoma and the NCX is a potential drug target for cancer therapy.

Ruthenium(II)-diphosphine complexes containing acylthiourea ligands are effective against lung and breast cancers.

We have synthesized and characterized three new ruthenium(II) diphosphine complexes containing an acylthiourea ligand, with the general formula [Ru(DPEPhos)(O,S)(bipy)]PF6, where DPEPhos = bis(2-(diphenylphosphino)phenyl)ether, bipy = 2,2'-bipyridine, and O,S = N,N-dimethyl-N'-(benzoyl)thiourea (1), N,N-dimethyl-N'-(furoyl)thiourea (2), and N,N-dimethyl-N'-(thiophenyl)thiourea (3), by several physicochemical techniques. We evaluated the ruthenium complexes for their cytotoxicity against two human cancer cell lines, A549 (lung) and MDA-MB-231 (breast), and two corresponding lines of non-cancer cells, MRC-5 (lung) and MCF-10A (breast). All the complexes are cytotoxic against the cancer cell lines; the IC50 values lie in the micromolar range (0.07-0.70 µM). Ruthenium complex 1 is more selective (7 times more active) toward lung cancer cells (A549) than toward non-cancer cells (MRC-5) and is 160 times more cytotoxic than cisplatin against A549 cells. Investigations of the mechanism of action of complex 1 in A549 cells demonstrated that it inhibits colony formation and promotes cell cycle arrest in the G1 phase and apoptotic cell death. DNA binding studies revealed that complexes 1-3 interact with the biomolecule via minor grooves. These complexes also interact with human serum albumin (HSA) and have affinity for site I by hydrophobic forces. Therefore, this new class of ruthenium complexes can act as cytotoxic agents, mainly for lung cancer treatment.

The beneficial effect of salubrinal on neuroinflammation and neuronal loss in intranigral LPS-induced hemi-Parkinson disease model in rats.

OBJECTIVE: Endoplasmic reticulum stress (ERS) and neuroinflammation are triggers for neurodegenerative disorders. Salubrinal is a selective inhibitor of protein phosphatase 1 (PP1) complex involving dephosphorylation of phosphorylated eukaryotic initiation factor-2α (eIF2α), the key crucial pathway in the ERS. Therefore, this study assessed the effects of inhibition of the ERS with salubrinal in the intranigral hemi-Parkinson disease (PD) model. MATERIALS AND METHODS: Animals were treated with salubrinal for one week after the PD model was created by intranigral lipopolysaccharide (LPS) administration. Apomorphine-induced rotation, rotarod, cylinder, and pole tests were performed to evaluate behavioral changes. Proinflammatory cytokines and the expression level of the dual specificity protein phosphatase 2 (DUSP2), PP1, and p-eIF2α were evaluated. Nigral expression of inducible nitric oxide synthase (iNOS), nuclear factor kappaB (Nf-κB), and cyclooxygenase (COX)-2 was determined. Finally, tyrosine hydroxylase and caspase-3/ caspase-9 expressions were assessed by immunohistochemistry. RESULTS: Salubrinal reduced the motor impairments and dopamine-related behavioral deficiencies caused by the LPS. Salubrinal attenuated the LPS-induced increased levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, and salubrinal rescued the loss of TH expression and dopamine levels and prevented the caspase-3/9 increase in the substantial nigra (SN). LPS potently increased iNOS, Nf-κB, and COX-2 expression, but this effect was reduced after salubrinal treatment. Additionally, salubrinal attenuated the LPS-induced PP1 and DUSP2 increase. CONCLUSION: Our results reveal that salubrinal is attenuating several inflammatory mediators and thereby decreased the inflammatory effects of LPS in the neurons of the SN. Together this results in increased cellular survival and maintained integrity of SN. Taken together our data show the beneficial effects of inhibition of ERS to restrict neuroinflammatory progression and neuronal loss in a PD model.