Folic Acid Exerts Post-Ischemic Neuroprotection In Vitro Through HIF-1α Stabilization

The constant failure of single-target drug therapies for ischemic stroke necessitates the development of novel pleiotropic pharmacological treatment approaches, to effectively combat the aftermath of this devastating disorder. The major objective of our study involves a multi-target drug repurposing strategy to stabilize hypoxia-inducible factor-1 α (HIF-1α) via a structure-based screening approach to simultaneously inhibit its regulatory proteins, PHD2, FIH, and pVHL. Out of 1424 Food and Drug Administration (FDA)-approved drugs that were screened, folic acid (FA) emerged as the top hit and its binding potential to PHD2, FIH, and pVHL was further verified by re-docking, molecular dynamics (MD) simulation and by Drug Affinity Responsive Target Stability (DARTS) assay. HIF-1α stabilization by FA was demonstrated by the nuclear translocation and increased green fluorescence emission of HIF-1α using HIF1α-GFPSpark tag vector. Further, FA treatment enhanced the cell survival following oxygen glucose deprivation and its neuroprotective mechanism was elucidated by measuring the expression of BAX, NFE2L2, VEGF, and EPO genes in a time-dependent manner (5 and 11 h following FA treatment). VEGF and EPO expressions were significantly increased by 5.41- and 1.35-folds, respectively, whereas BAX expression reduced by 4-fold at 11 h post-FA treatment. NFE2L2 expression was elevated (1.65-fold) at 5 h with no major difference at 11 h post-FA treatment. The chicken chorioallantoic membrane (CAM) assay demonstrated the pro-angiogenic potential of FA as evidenced by an increased blood vessel density and branching. The present study elucidates for the first time that the post-ischemic neuroprotection exerted by FA may be attributed to its HIF-1α stabilization and pro-angiogenic properties.     

The Synergistic In Vitro and In Vivo Antitumor Effect of Combination Therapy with Salinomycin and 5-Fluorouracil against Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the few cancers in which a continuous increase in incidence has been observed over several years. Drug resistance is a major problem in the treatment of HCC. In the present study, we used salinomycin (Sal) and 5-fluorouracil (5-FU) combination therapy on HCC cell lines Huh7, LM3 and SMMC-7721 and nude mice subcutaneously tumor model to study whether Sal could increase the sensitivity of hepatoma cells to the traditional chemotherapeutic agent such as 5-FU. The combination of Sal and 5-FU resulted in a synergistic antitumor effect against liver tumors both in vitro and in vivo. Sal reversed the 5-FU-induced increase in CD133(+) EPCAM(+) cells, epithelial–mesenchymal transition and activation of the Wnt/β-catenin signaling pathway. The combination of Sal and 5-FU may provide us with a new approach to reverse drug resistant for the treatment of patients with HCC.     

Combination of Gefitinib and DNA Methylation Inhibitor Decitabine Exerts Synergistic Anti-Cancer Activity in Colon Cancer Cells

Despite recent advances in the treatment of human colon cancer, the chemotherapy efficacy against colon cancer is still unsatisfactory. In the present study, effects of concomitant inhibition of the epidermal growth factor receptor (EGFR) and DNA methyltransferase were examined in human colon cancer cells. We demonstrated that decitabine (a DNA methyltransferase inhibitor) synergized with gefitinib (an EGFR inhibitor) to reduce cell viability and colony formation in SW1116 and LOVO cells. However, the combination of the two compounds displayed minimal toxicity to NCM460 cells, a normal human colon mucosal epithelial cell line. The combination was also more effective at inhibiting the AKT/mTOR/S6 kinase pathway. In addition, the combination of decitabine with gefitinib markedly inhibited colon cancer cell migration. Furthermore, gefitinib synergistically enhanced decitabine-induced cytotoxicity was primarily due to apoptosis as shown by Annexin V labeling that was attenuated by z-VAD-fmk, a pan caspase inhibitor. Concomitantly, cell apoptosis resulting from the co-treatment of gefitinib and decitabine was accompanied by induction of BAX, cleaved caspase 3 and cleaved PARP, along with reduction of Bcl-2 compared to treatment with either drug alone. Interestingly, combined treatment with these two drugs increased the expression of XIAP-associated factor 1 (XAF1) which play an important role in cell apoptosis. Moreover, small interfering RNA (siRNA) depletion of XAF1 significantly attenuated colon cancer cells apoptosis induced by the combination of the two drugs. Our findings suggested that gefitinib in combination with decitabine exerted enhanced cell apoptosis in colon cancer cells were involved in mitochondrial-mediated pathway and induction of XAF1 expression. In conclusion, based on the observations from our study, we suggested that the combined administration of these two drugs might be considered as a novel therapeutic regimen for treating colon cancer.     

Neuroprotection by the N-Methyl-d-Aspartate Receptor Antagonist CGP 40116: In Vivo and In Vitro Studies

Abstract: The goal of this study was to evaluate the effects of a novel competitive N -methyl- d -aspartate (NMDA) receptor antagonist, d -( E )-2-amino-4-methyl-5-phosphono-3-pentoic acid (CGP 40116), on neuronal damage in vivo and in vitro. We studied 20 rabbits that underwent a 2-h occlusion of the left internal carotid, anterior cerebral, and middle cerebral arteries followed by 4 h of reperfusion. Ten minutes after occlusion the animals were treated with either normal saline (n = 7) or CGP 40116 at two different doses (20 mg/kg, n = 6; 40 mg/kg, n = 7) administered over a 5-min period. Somatosensory evoked potentials were used to confirm adequate ischemia and neuronal injury was assessed by histopathology and magnetic resonance imaging. CGP 40116 decreased cortical ischemic neuronal damage by 74 and 77% (control, 37.8%± 13.1%; CGP 20 mg/kg, 9.9 ± 3.6%; CGP 40 mg/kg, 8.7 ± 3.7%; p < 0.01) and reduced cortical ischemic edema by 52 and 35% (control, 42.3 ± 10.4%; CGP 20 mg/kg, 20.1 ± 6.7%; CGP 40 mg/kg, 27.5 ± 13.3%; p < 0.05) but did not protect against striatal injury. We performed a second study using primary cell cultures from mouse neocortex to determine the effects of CGP 40116 on neuronal death induced by a 10-min exposure to 500 µ M NMDA or by 45 min of oxygen-glucose deprivation (OGD). Our results demonstrate that CGP 40116 was effective at attenuating neuronal death in a concentration-dependent manner (ED₅₀ of 3.2 µ M against NMDA toxicity and 23.1 µ M against OGD) as measured by lactate dehydrogenase levels 24 h after the insult. The neuroprotective effects of CGP 40116 in vivo and in vitro suggest it may be of potential clinical therapeutic value.     

In Vitro Synergistic Activities of Essential Oils and Surfactants in Combination with Cosmetic Preservatives Against Pseudomonas aeruginosa and Staphylococcus aureus

The aim of this study is to evaluate possible synergistic antimicrobial interactions between common cosmetic preservatives and selected essential oils or surfactants. The antimicrobial efficacy of six essential oils, three surfactants and five preservatives against Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 43387 was assessed by a broth micro-dilution assay. MICs for individual and combined antimicrobials were determined and then transformed to fractional inhibitory concentration (FIC) indexes. All essential oils exhibited antibacterial activity; among surfactants, bacteria resulted most susceptible to the cationic agent. Synergy was observed when essential oils of eucalyptus and mint were combined with methylparaben against P. aeruginosa, while essential oils of mint, oregano and sage combined with propylparaben and imidazolidinyl urea acted against S. aureus. Many binary mixtures of preservatives and surfactants produced synergistic activity with the most effective interactions involving the cationic and amphoteric compounds under study. FIC indexes demonstrated synergistic effects when preservatives were combined with either essential oils or surfactants against both bacterial strains. These results highlight the potential usefulness of essential oils and surfactants to enhance the activities of conventional biocides. This kind of study should contribute to the selection and optimization of preservative systems for cosmetic preparations.     

Activation of Neuropeptide Y Receptors Modulates Retinal Ganglion Cell Physiology and Exerts Neuroprotective Actions In Vitro

Neuropeptide Y (NPY) is expressed in mammalian retina but the location and potential modulatory effects of NPY receptor activation remain largely unknown. Retinal ganglion cell (RGC) death is a hallmark of several retinal degenerative diseases, particularly glaucoma. Using purified RGCs and ex vivo rat retinal preparations, we have measured RGC intracellular free calcium concentration ([Ca²⁺]_i) and RGC spiking activity, respectively. We found that NPY attenuated the increase in the [Ca²⁺]_i triggered by glutamate mainly via Y₁ receptor activation. Moreover, (Leu³¹, Pro³⁴)−NPY, a Y₁/Y₅ receptor agonist, increased the initial burst response of OFF-type RGCs, although no effect was observed on RGC spontaneous spiking activity. The Y₁ receptor activation was also able to directly modulate RGC responses by attenuating the NMDA-induced increase in RGC spiking activity. These results suggest that Y₁ receptor activation, at the level of inner or outer plexiform layers, leads to modulation of RGC receptive field properties. Using in vitro cultures of rat retinal explants exposed to NMDA, we found that NPY pretreatment prevented NMDA-induced cell death. However, in an animal model of retinal ischemia-reperfusion injury, pretreatment with NPY or (Leu³¹, Pro³⁴)−NPY was not able to prevent apoptosis or rescue RGCs. In conclusion, we found modulatory effects of NPY application that for the first time were detected at the level of RGCs. However, further studies are needed to evaluate whether NPY neuroprotective actions detected in retinal explants can be translated into animal models of retinal degenerative diseases.     

A Novel Compound NSC745885 Exerts an Anti-Tumor Effect on Tongue Cancer SAS Cells In Vitro and In Vivo

Objective

Oral squamous cell carcinoma (OSCC) is a prevalent cancer, especially in developing countries. Anthracyclines and their anthraquinone derivatives, such as doxorubicin, exhibit a cell growth inhibitory effect and have been used as anti-cancer drugs for many years. However, the cardiotoxicity of anthracycline antibiotics is a major concern in their clinical application. NSC745885 is a novel compound synthesized from 1,2-diaminoanthraquinone, which subsequently reacts with thionyl chloride and triethylamine. The present study aimed to investigate the anti-oral cancer potential and the safety of NSC745885.

Methods

We investigated the anti-cancer potential of NSC745885 in oral squamous carcinoma cell lines and in an in vivo oral cancer xenograft mouse model. The expression of apoptotic related genes were evaluated by real-time RT-PCR and western bloting, and the in vivo assessment of apoptotic marker were measured by immunohistochemical staining. The anti-tumor efficiency and safety between doxorubicin and NSC745885 were also compared.

Results

Our results demonstrated that NSC745885 exhibits anti-oral cancer activity through the induction of apoptosis in cancer cells and in tumor-bearing mice, and this treatment did not induce marked toxicity in experimental mice. This compound also exhibits a comparable anti-tumor efficiency and a higher safety in experimental mice when compared to doxorubicin.

Conclusions

The data of this study provide evidence for NSC745885 as a potential novel therapeutic drug for the treatment of human OSCC.     

Calcitriol Imparts Neuroprotection In Vitro to Midbrain Dopaminergic Neurons by Upregulating GDNF Expression

During development a tightly controlled signaling cascade dictates the differentiation, maturation and survival of developing neurons. Understanding this signaling mechanism is important for developing therapies for neurodegenerative illnesses. In previous work we have sought to understand the complex signaling pathways responsible for the development of midbrain dopamine neurons using a proteomic approach. One protein we have identified as being expressed in developing midbrain tissue is the vitamin D receptor. Therefore we investigated the effect of the biologically active vitamin D₃ metabolite, calcitriol, on primary fetal ventral mesencephalic cultures of dopamine neurons. We observed a dose responsive increase in numbers of rat primary dopamine neurons when calcitriol was added to culture media. Western blot data showed that calcitriol upregulated the expression of glial derived neurotrophic factor (GDNF). Blocking GDNF signaling could prevent calcitriol’s ability to increase numbers of dopamine neurons. An apoptosis assay and cell birth dating experiment revealed that calcitriol increases the number of dopamine neurons through neuroprotection and not increased differentiation. This could have implications for future neuroprotective PD therapies.     

In Vitro Synergistic Activities of the Hybrid Antimicrobial Peptide MelitAP-27 in Combination with Conventional Antibiotics Against Planktonic and Biofilm Forming Bacteria

The extensive use of antibiotics for the treatment of human infections during the last few decades has led to a dramatic increase in the emergence of multidrug-resistant bacteria (MDRB) among various bacterial strains. Global research is currently focused on finding novel alternative agents with different mechanisms of action rather than the use of conventional antibiotics to counteract the threat of bacterial and biofilm infections. Antimicrobial peptides represent promising alternative agents for conventional antibiotics as these molecules display a broad spectrum of activity against several microorganisms. Recently, we have designed a novel hybrid antimicrobial peptide named MelitAP-27. This peptide has been found to display potent broad spectrum and selective in vitro antimicrobial activities against a wide range of Gram-positive and Gram-negative bacteria. In the present study, the in vitro antimicrobial and antibiofilm activities of the peptide alone and in combination with five different types of antibiotics were assessed against wild-type and resistant Gram-positive and Gram-negative bacterial strains. Our results showed that most of the combination groups displayed a synergistic mode of action against planktonic and biofilm forming bacteria which resulted in decreasing the effective MIC values for MelitAP-27 to the nanomolar concentrations. These effective concentrations were associated with negligible toxicities on mammalian cells. The results of our study indicate that combinations of MelitAP-27 with conventional antibiotics may be pursued as a potential novel treatment strategy against MDRB and biofilm forming bacteria.     

Combination Treatment of Hypothermia and Mesenchymal Stromal Cells Amplifies Neuroprotection in Primary Rat Neurons Exposed to Hypoxic-Ischemic-Like Injury In Vitro: Role of the Opioid System

This study was designed to reveal the therapeutic regimen and mechanism of action underlying hypothermia treatment in combination with stem cell transplantation for ameliorating neonatal hypoxic-ischemic-like injury. Primary rat neurons were exposed to oxygen-glucose deprivation (OGD), which produced hypoxic-ischemic-like injury in vitro, then incubated at 25°C (severe hypothermia), 34°C (moderate hypothermia), and 37°C (normothermia) with or without subsequent co-culture with mesenchymal stromal cells (MSCs). Combination treatment of moderate hypothermia and MSCs significantly improved cell survival and mitochondrial activity after OGD exposure. The exposure of delta opioid human embryonic kidney cells (HEK293) to moderate hypothermia attenuated OGD-mediated cell alterations, which were much more pronounced in HEK293 cells overexpressing the delta opioid receptor. Further, the addition of delta opioid peptide to 34°C hypothermia and stem cell treatment in primary rat neurons showed synergistic neuroprotective effects against OGD which were significantly more robust than the dual combination of moderate hypothermia and MSCs, and were significantly reduced, but not completely abolished, by the opioid receptor antagonist naltrexone altogether implicating a ligand-receptor mechanism of neuroprotection. Further investigations into non-opioid therapeutic signaling pathways revealed growth factor mediation and anti-apoptotic function accompanying the observed therapeutic benefits. These results support combination therapy of hypothermia and stem cells for hypoxic-ischemic-like injury in vitro, which may have a direct impact on current clinical trials using stand-alone hypothermia or stem cells for treating neonatal encephalopathy.     

In Vitro Activity of Rifampicin and Verapamil Combination in Multidrug-Resistant Mycobacterium tuberculosis

The aim of the present study was to evaluate the effect of the combination of rifampicin (RIF) and verapamil (VP) against the Mycobacterium tuberculosis H₃₇Rv reference strain and six multidrug-resistant (MDR) M. tuberculosis clinical isolates by determining Time-Kill Curves and the ability to efflux drug by fluorometry. The RIF+VP combination showed synergism in one MDR clinical isolate. For the other five MDR clinical isolates, the drug combination showed no interaction. The MDR clinical isolate had lower ethidium bromide (EtBr) accumulation when exposed to the RIF+VP combination, compared with RIF and VP exposure alone. The other MDR clinical isolates showed no significant difference in EtBr accumulation. These results suggest greater efflux action in one of the MDR clinical isolates compared with the M. tuberculosis H₃₇Rv reference strain. The other five MDR isolates may have additional mechanisms of drug resistance to RIF. The use of the RIF+VP combination made one MDR bacillus more susceptible to RIF probably by inhibiting efflux pumps, and this combination therapy, in some cases, may contribute to a reduction of resistance to RIF in M. tuberculosis.     

Tribulusterine Containing Tribulus terrestris Extract Exhibited Neuroprotection Through Attenuating Stress Kinases Mediated Inflammatory Mechanism: In Vitro and In Vivo Studies

The present study has been aimed to explore the different secondary messengers of the inflammatory pathway NF-κB, kinases (JNK, P38MAPK, GSK3β/βcatenin), apoptosis pathway (Caspase-3 and AIF), and neuronal survival pathway (BDNF) in order to understand the neuroprotective mechanism of aqueous extract of Tribulus terrestris (AQTT). In primary cortical neurons, the ischemic condition was induced through oxygen–glucose deprivation (OGD). Anti-inflammatory activity of AQTT was evaluated in formalin induced inflammation model and carrageenan-induced paw edema test. The bilateral common carotid artery occlusion model was employed for whole animal studies. Treatment of AQTT (100 mg/kg) significantly reduced the inflammation induced by formalin and carrageenan. The neuroprotective mechanism of AQTT (50 and 100 mg/kg) was assessed by pre- and post-administration. The results indicate down regulation of kinases and NFkB, suggesting possible anti-inflammatory activity of AQTT. Additionally, AQTT down regulated both caspase dependent and independent apoptotic pathways suggesting its possible anti-apoptotic activity. The treatment of AQTT also reduced GSK3β levels and increased p-Ser9 GSK3β levels; stabilizing the unphosphorylated form of β-catenin and its translocation into the nucleus suggesting role of AQTT in neuronal survival and GSK3β mediated anti-inflammatory property. In comparison to pretreatment, post treatment of AQTT had lesser effects indicating tribulusterine standardized AQTT may have prophylactic effect. This study can be concluded with the thesis that AQTT has neuroprotective effect through alternating neuroinflammation, apoptosis, and promoting neuron survival. Being that it produced better effect with pretreatment, exploring this with thrombolytic drugs will be beneficial. For the first time AQTT has been reported for this indication.

     

Upregulating Noxa by ER Stress,Celastrol Exerts Synergistic Anti-Cancer Activity in Combination with ABT-737 in Human Hepatocellular Carcinoma Cells

The human hepatocellular carcinoma (HCC) represents biologically aggressive and chemo-resistant cancers. Owing to the low affinity with the apoptotic factor Mcl-1, the BH3 mimetic drug ABT-737 failed to exert potent cancer-killing activities in variety of cancer models including HCC. The current study demonstrated that combining ABT-737 and Celastrol synergistically suppressed HCC cell proliferation, and induced apoptosis which was accompanied with the activation of caspase cascade and release of cytochrome c from mitochondria. Further study revealed that the enhanced Noxa caused by Celastrol was the key factor for the synergy, since small interfering RNA-mediated knockdown of Noxa expression in HCC cells resulted in decreased apoptosis and attenuated anti-proliferative effects of the combination. In addition, our study unraveled that, upon Celastrol exposure, the activation of endoplasmic reticulum (ER) stress, specifically, the eIF2α-ATF4 pathway played indispensable roles in the activation of Noxa, which was validated by the observation that depletion of ATF4 significantly abrogated the Noxa elevation by Celastrol. Our findings highlight a novel signaling pathway through which Celastrol increase Noxa expression, and suggest the potential use of ATF4-mediated regulation of Noxa as a promising strategy to improve the anti-cancer activities of ABT-737.     

Combination of Bioactive Polymeric Membranes and Stem Cells for Periodontal Regeneration: In Vitro and In Vivo Analyses

Regeneration of periodontal tissues requires a concerted effort to obtain consistent and predictable results in vivo. The aim of the present study was to test a new family of bioactive polymeric membranes in combination with stem cell therapy for periodontal regeneration. In particular, the novel polyester poly(isosorbide succinate-co-L-lactide) (PisPLLA) was compared with poly(L-lactide) (PLLA). Both polymers were combined with collagen (COL), hydroxyapatite (HA) and the growth factor bone morphogenetic protein-7 (BMP7), and their osteoinductive capacity was evaluated via in vitro and in vivo experiments. Membranes composed of PLLA/COL/HA or PisPLLA/COL/HA were able to promote periodontal regeneration and new bone formation in fenestration defects in rat jaws. According to quantitative real-time polymerase chain reaction (qRT-PCR) and Alizarin Red assays, better osteoconductive capacity and increased extracellular mineralization were observed for PLLA/COL/HA, whereas better osteoinductive properties were associated with PisPLLA/COL/HA. We concluded that membranes composed of either PisPLLA/COL/HA or PLLA/COL/HA present promising results in vitro as well as in vivo and that these materials could be potentially applied in periodontal regeneration.     

Synergistic Effect of Selenium and Melatonin on Neuroprotection in Cerebral Ischemia in Rats

The synergistic scavenger effects of selenium and melatonin collectively we called Se-Mel was studied on the prevention of neuronal injury induced by ischemia/reperfusion. Male Wistar rats were treated with sodium selenite (0.1 mg/kg, i.p.) and melatonin (10 mg/kg, i.p.) 30 min before the middle carotid artery occlusion (MCAO) and immediately after MCAO to male Wistar rats and was continued for 3 days once daily at the interval of 24 h. Behavioral activity (spontaneous motor activity and motor deficit) was improved in Se-Mel-treated rats as compared to MCAO group rats. The level of glutathione and the activity of antioxidant enzymes was depleted significantly while the content of thiobarbituric acid reactive substances, protein carbonyl, and nitric oxide radical (NO^·) was increased significantly in MCAO group. Systemic administration of Se-Mel ameliorated oxidative stress and improves ischemia/reperfusion-induced focal cerebral ischemia. Se-Mel also inhibited inducible nitric oxide synthase expression in Se-Mel+MCAO group as compared to MCAO group rats. Thus, Se-Mel has shown an excellent neuroprotective effect against ischemia/reperfusion injury through an anti-ischemic pathway. In conclusion, we demonstrated that the pretreatment with Se-Mel at the onset of reperfusion, reduced post-ischemic damage, and improved neurological outcome following transient focal cerebral ischemia in male Wistar rat.     

In Vitro Susceptibility of Pseudomonas aeruginosa to Carbenicillin and the Combination of Carbenicillin and Gentamicin

One hundred and eleven strains of Pseudomonas aeruginosa isolated from clinical material were studied for susceptibility to carbenicillin. Of the strains, 80% were inhibited by 125 mug/ml or more. The combination of carbenicillin and gentamicin was shown to have inhibitory and bactericidal synergistic effect on 15 of 16 strains of P. aeruginosa tested. There was poor correlation between the single-disc sensitivity method and the tube dilution method.     

Posaconazole Exhibits In Vitro and In Vivo Synergistic Antifungal Activity with Caspofungin or FK506 against Candida albicans

The object of this study was to test whether posaconazole, a broad-spectrum antifungal agent inhibiting ergosterol biosynthesis, exhibits synergy with the β-1,3 glucan synthase inhibitor caspofungin or the calcineurin inhibitor FK506 against the human fungal pathogen Candida albicans. Although current drug treatments for Candida infection are often efficacious, the available antifungal armamentarium may not be keeping pace with the increasing incidence of drug resistant strains. The development of drug combinations or novel antifungal drugs to address emerging drug resistance is therefore of general importance. Combination drug therapies are employed to treat patients with HIV, cancer, or tuberculosis, and has considerable promise in the treatment of fungal infections like cryptococcal meningitis and C. albicans infections. Our studies reported here demonstrate that posaconazole exhibits in vitro synergy with caspofungin or FK506 against drug susceptible or resistant C. albicans strains. Furthermore, these combinations also show in vivo synergy against C. albicans strain SC5314 and its derived echinocandin-resistant mutants, which harbor an S645Y mutation in the CaFks1 β-1,3 glucan synthase drug target, suggesting potential therapeutic applicability for these combinations in the future.