Tamoxifen enhances erlotinib-induced cytotoxicity through down-regulating AKT-mediated thymidine phosphorylase expression in human non-small-cell lung cancer cells

Tamoxifen is a triphenylethylene nonsteroidal estrogen receptor (ER) antagonist used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Thymidine phosphorylase (TP) is an enzyme of the pyrimidine salvage pathway which is upregulated in cancers. In this study, tamoxifen treatment inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with tamoxifen decreased TP mRNA and protein levels through AKT inactivation. Furthermore, expression of constitutively active AKT (AKT-CA) vectors significantly rescued the decreased TP protein and mRNA levels in tamoxifen-treated NSCLC cells. In contrast, combination treatment with PI3K inhibitors (LY294002 or wortmannin) and tamoxifen further decreased the TP expression and cell viability of NSCLC cells. Knocking down TP expression by transfection with small interfering RNA of TP enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Erlotinib (Tarceva, OSI-774), an orally available small molecular inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for clinical treatment of NSCLC. Compared to a single agent alone, tamoxifen combined with erlotinib resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-AKT and phospho-ERK1/2, and reduced TP protein levels. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and erlotinib for the treatment of NSCLC.     

Protective role of amantadine in mitochondrial dysfunction and oxidative stress mediated by hepatitis C virus protein expression

Amantadine is an antiviral and antiparkinsonian drug that has been evaluated in combination therapies against hepatitis C virus (HCV) infection. Controversial results have been reported concerning its efficacy, and its mechanism of action remains unclear. Data obtained in vitro suggested a role of amantadine in inhibiting HCV p7-mediated cation conductance. In keeping with the fact that mitochondria are responsible to ionic fluxes and that HCV infection impairs mitochondrial function, we investigated a potential role of amantadine in modulating mitochondrial function. Using a well-characterized inducible cell line expressing the full-length HCV polyprotein, we found that amantadine not only prevented but also rescued HCV protein-mediated mitochondrial dysfunction. Specifically, amantadine corrected (i) overload of mitochondrial Ca²⁺; (ii) inhibition of respiratory chain activity and oxidative phosphorylation; (iii) reduction of membrane potential; and (iv) overproduction of reactive oxygen species. The effects of amantadine were observed within 15 min following drug administration and confirmed in Huh-7.5 cells transfected with an infectious HCV genome. These effects were also observed in cells expressing subgenomic HCV constructs, indicating that they are not mediated or only in part mediated by p7. Single organelle analyzes carried out on isolated mouse liver mitochondria demonstrated that amantadine induces hyperpolarization of the membrane potential. Moreover, amantadine treatment increased the calcium threshold required to trigger mitochondrial permeability transition opening. In conclusion, these results support a role of amantadine in preserving cellular bioenergetics and redox homeostasis in HCV-infected cells and unveil an effect of the drug which might be exploited for a broader therapeutic utilization.     

Functional G-protein-coupled receptor 35 is expressed by neurons in the CA1 field of the hippocampus

The G-protein-coupled receptor 35 (GPR35) was de-orphanized after the discovery that kynurenic acid (KYNA), an endogenous tryptophan metabolite, acts as an agonist of this receptor. Abundant evidence supports that GPR35 exists primarily in peripheral tissues. Here, we tested the hypothesis that GPR35 exists in the hippocampus and influences the neuronal activity. Fluorescence immunohistochemical staining using an antibody anti-NeuN (a neuronal marker), an antibody anti-GFAP (a glial marker), and an antibody anti-GPR35 revealed that neurons in the stratum oriens, stratum pyramidale, and stratum radiatum of the CA1 field of the hippocampus express GPR35. To determine the presence of functional GPR35 in the neurocircuitry, we tested the effects of various GPR35 agonists on the frequency of spontaneous action potentials recorded as fast current transients (CTs) from stratum radiatum interneurons (SRIs) under cell-attached configuration in rat hippocampal slices. Bath application of the GPR35 agonists zaprinast (1–10 μM), dicumarol (50–100 μM), pamoic acid (500–1000 μM), and amlexanox (3 μM) produced a concentration- and time-dependent reduction in the frequency of CTs. Superfusion of the hippocampal slices with the GPR35 antagonist ML145 (1 μM) increased the frequency of CTs and reduced the inhibitory effect of zaprinast. Bath application of phosphodiesterase 5 inhibitor sildenafil (1 or 5 μM) was ineffective, whereas a subsequent application of zaprinast was effective in reducing the CT frequency. The present results demonstrate for the first time that functional GPR35s are expressed by CA1 neurons and suggest that these receptors can be molecular targets for controlling neuronal activity in the hippocampus.     

Therapeutic approaches against common structural features of toxic oligomers shared by multiple amyloidogenic proteins

Impaired proteostasis is one of the main features of all amyloid diseases, which are associated with the formation of insoluble aggregates from amyloidogenic proteins. The aggregation process can be caused by overproduction or poor clearance of these proteins. However, numerous reports suggest that amyloid oligomers are the most toxic species, rather than insoluble fibrillar material, in Alzheimer's, Parkinson's, and Prion diseases, among others. Although the exact protein that aggregates varies between amyloid disorders, they all share common structural features that can be used as therapeutic targets. In this review, we focus on therapeutic approaches against shared features of toxic oligomeric structures and future directions.     

JAK/STAT途径调节瘦素诱导的肝星状细胞Ⅰ型胶原基因的表达

目的研究瘦素对肝星状细胞(HSC)α1(Ⅰ)型胶原mRNA表达和蛋白合成的影响,探讨Janus激酶/信号传导及活化转录因子(JAK/STAT)信号传导通路在瘦素诱导的HSC胶原基因表达过程中的作用。方法采用RT-PCR法、ELISA法和Western blot法分别检测不同浓度的瘦素对人肝星状细胞株LX-2α1(Ⅰ)型胶原mRNA表达、蛋白合成以及JAK1、STAT3磷酸化状态的影响;采用Western blot法和RT-PCR法分别观察JAK1抑制剂AG490对瘦素诱导的JAK1磷酸化和α1(Ⅰ)型胶原mRNA表达的影响;采用Western blot法分别观察AG490、LX-2转染STAT3反义寡核苷酸(STAT3-ASON)对瘦素诱导的STAT3磷酸化状态的影响;应用RT-PCR法检测LX-2转染STAT3-ASON对瘦素诱导的α1(Ⅰ)型胶原mRNA表达的影响。结果瘦素增加LX-2α1(Ⅰ)型胶原mRNA表达和蛋白合成,呈剂量效应关系,当瘦素浓度为80μg·L-1时达到最大值;瘦素促进JAK1、STAT3磷酸化,呈时间效应关系;AG490完全阻断瘦素诱导的JAK1、STAT3磷酸化和α1(Ⅰ)型胶原mRNA的表达;LX-2转染STAT3-ASON阻断瘦素诱导的STAT3磷酸化和α1(Ⅰ)型胶原mRNA的表达。结论瘦素通过增加HSCα1(Ⅰ)型胶原mRNA表达和蛋白合成而在肝纤维化发生发展过程中发挥重要的作用,JAK/STAT信号传导通路参与并调节该过程,AG490和LX-2转染STAT3-ASON可有效阻滞此传导途径。在人HSC中,活化的JAK1和STAT3信号可作为肝纤维化治疗新的分子靶点。     

Chronic oleoylethanolamide treatment improves spatial cognitive deficits through enhancing hippocampal neurogenesis after transient focal cerebral ischemia

Oleoylethanolamide (OEA) has been shown to have neuroprotective effects after acute cerebral ischemic injury. The aim of this study was to investigate the effects of chronic OEA treatment on ischemia-induced spatial cognitive impairments, electrophysiology behavior and hippocampal neurogenesis. Daily treatments of 30 mg/kg OEA significantly ameliorated spatial cognitive deficits and attenuated the inhibition of long-term potentiation (LTP) in the middle cerebral artery occlusion (MCAO) rat model. Moreover, OEA administration improved cognitive function in a manner associated with enhanced neurogenesis in the hippocampus. Further study demonstrated that treatment with OEA markedly increased the expressions of brain-derived neurotrophic factor (BDNF) and peroxisome proliferator-activated receptors α (PPARα). Our data suggest that chronic OEA treatment can exert functional recovery of cognitive impairments and neuroprotective effects against cerebral ischemic insult in rats via triggering of neurogenesis in the hippocampus, which supports the therapeutic use of OEA for cerebral ischemia.     

Angiotensin (1–7) protects against stress-induced gastric lesions in rats

Stress ulcers can develop with severe physiological stress, and have been proposed as being brain-driven events. New findings continue to suggest that stress ulcers can be more effectively managed through central manipulation rather than by simply altering local gastric factors. Angiotensin (1–7) (Ang (1–7)) is present as an endogenous constituent of the brain and stomach. The beneficial effects of Ang (1–7) have been confirmed in the vessels, brain, heart, kidney, liver and lungs, but not in the stomach. Given the accumulating evidence suggesting the anti-stress activities of Ang (1–7), its potential gastroprotective effect in the context of stress requires further investigation. In the present study, rat gastric mucosal lesions were induced by 2 h of cold-restraint stress. We observed that these lesions were significantly attenuated after 1 week of intracerebroventricular treatment with Ang (1–7). This gastroprotective effect was associated with attenuated oxidative stress and suppressed acid secretion. Brain Ang (1–7) administration profoundly modified responses to stress, indicated by altered levels of several stress hormones, including Ang II, glucocorticoid, norepinephrine, serotonin, and dopamine, in blood or stress-related brain regions. These findings indicate that Ang (1–7) exerts anti-stress activities by restoring the gastric microenvironment and modulating the stress pathways. Ang (1–7) may be a promising agent for stress ulcer prophylaxis and therapy, administered through brain-permeable mimics or carriers.     

The role of JAK/STAT signaling pathway and its inhibitors in diseases

The JAK/STAT signaling pathway is an universally expressed intracellular signal transduction pathway and involved in many crucial biological processes, including cell proliferation, differentiation, apoptosis, and immune regulation. It provides a direct mechanism for extracellular factors-regulated gene expression. Current researches on this pathway have been focusing on the inflammatory and neoplastic diseases and related drug.The mechanism of JAK/STAT signaling is relatively simple. However, the biological consequences of the pathway are complicated due to its crosstalk with other signaling pathways. In addition, there is increasing evidence indicates that the persistent activation of JAK/STAT signaling pathway is closely related to many immune and inflammatory diseases, yet the specific mechanism remains unclear. Therefore, it is necessary to study the detailed mechanisms of JAK/STAT signaling in disease formation to provide critical reference for clinical treatments of the diseases.In this review, we focus on the structure of JAKs and STATs, the JAK/STAT signaling pathway and its negative regulators, the associated diseases, and the JAK inhibitors for the clinical therapy.     

Dancing with chemical formulae of antivirals: A panoramic view (Part 2)

In this second part of “Dancing with antivirals as chemical formulae” I will focus on a number of chemical compounds that in the last few years have elicited more than common attraction from a commercial viewpoint: (i) favipiravir (T-705), as it is active against influenza, but also several other RNA viruses; (ii) neuraminidase inhibitors such as zanamivir and oseltamivir; (iii) peramivir and laninamivir octanoate, which might be effective against influenza virus following a single (intravenous or inhalation) administration; (iv) sofosbuvir, the (anticipated) cornerstone for the interferon-free therapy of HCV infections; (v) combinations of DAAs (direct antiviral agents) to achieve, in no time, a sustained virus response (SVR) against HCV infection; (vi) HIV protease inhibitors, the latest and most promising being darunavir; (vii) the integrase inhibitors (INIs) (raltegravir, elvitegravir, dolutegravir), representing a new dimension in the anti-HIV armamentarium; (viii), a new class of helicase primase inhibitors (HPIs) that may exceed acyclovir and the other anti-herpes compounds in both potency and safety; (ix) CMX-001, as the latest of Dr. Antonín Holý’s legacy for its activity against poxviruses and CMV infections, and (x) noroviruses for which the ideal antiviral compounds are still awaited for.     

Mitochondrial dysfunction in cancer chemoresistance

Mitochondrial dysfunction has been associated with cancer development and progression. Recent evidences suggest that pathogenic mutations or depletion of the mitochondrial genome can contribute to development of chemoresistance in malignant tumors. In this review we will describe the current knowledge on the role of mitochondrial dysfunction in the development of chemoresistance in cancer. We will also discuss the significance of this research topic in the context of development of more effective, targeted therapeutic modalities and diagnostic strategies for cancer patients, with a particular focus on the potential use of PARP inhibitors in cancer patients displaying mitochondrial DNA mutations. We will discuss recent studies highlighting the importance of the cross-talk between the tumor microenvironment and mitochondrial functionality in determining selective response to certain chemotherapeutic drugs. Finally, owing to the similarities between cancer and yeast cell metabolism, we will point out the use of yeast as a model system to study cancer-related genes and for anti-cancer drugs screening.     

Internal mouthpiece designs as a future perspective for enhanced aerosol deposition. Comparative results for aerosol chemotherapy and aerosol antibiotics

Background

In an effort to identify factors producing a finest mist from Jet-Nebulizers we designed 2 mouthpieces with 4 different internal designs and 1–3 compartments.

Materials and methods

Ten different drugs previous used with their “ideal” combination of jet-nebulizer, residual-cup and loading were used. For each drug the mass median aerodynamic diameter size had been established along with their “ideal” combination.

Results

For both mouthpiece, drug was the most important factor due the high F-values (F_large = 251.7, p < 0.001 and F_small = 60.1, p < 0.001) produced. The design affected the droplet size but only for large mouthpiece (F_large = 5.99, p = 0.001, F_small = 1.72, p = 0.178). Cross designs create the smallest droplets (2.271) so differing from the other designs whose mean droplets were greater and equal ranging between 2.39 and 2.447. The number of compartments in the two devices regarding the 10 drugs was found not statistically significant (p-values 0.768 and 0.532 respectively). Interaction effects between drugs and design were statistically significant for both devices (F_large = 8.87, p < 0.001, F_small = 5.33, p < 0.001).

Conclusion

Based on our experiment we conclude that further improvement of the drugs intended for aerosol production is needed. In addition, the mouthpiece design and size play an important role in further enhancing the fine mist production and therefore further experimentation is needed.     

Correlating FAAH and anandamide cellular uptake inhibition using N-alkylcarbamate inhibitors: From ultrapotent to hyperpotent

Besides the suggested role of a putative endocannabinoid membrane transporter mediating the cellular uptake of the endocannabinoid anandamide (AEA), this process is intrinsically coupled to AEA degradation by the fatty acid amide hydrolase (FAAH). Differential blockage of each mechanism is possible using specific small-molecule inhibitors. Starting from the natural product-derived 2E,4E-dodecadiene scaffold previously shown to interact with the endocannabinoid system (ECS), a series of diverse N-alkylcarbamates were prepared with the aim of generating novel ECS modulators. While being inactive at cannabinoid receptors and monoacylglycerol lipase, these N-alkylcarbamates showed potent to ultrapotent picomolar FAAH inhibition in U937 cells. Overall, a highly significant correlation (Spearman's rho = 0.91) was found between the inhibition of FAAH and AEA cellular uptake among 54 compounds. Accordingly, in HMC-1 cells lacking FAAH expression the effect on AEA cellular uptake was dramatically reduced. Unexpectedly, 3-(4,5-dihydrothiazol-2-yl)phenyl carbamates and the 3-(1,2,3-thiadiazol-4-yl)phenyl carbamates WOBE490, WOBE491 and WOBE492 showed a potentiation of cellular AEA uptake inhibition in U937 cells, resulting in unprecedented femtomolar (hyperpotent) IC₅₀ values. Potential methodological issues and the role of cellular accumulation of selected probes were investigated. It is shown that albumin impacts the potency of specific N-alkylcarbamates and, more importantly, that accumulation of FAAH inhibitors can significantly increase their effect on cellular AEA uptake. Taken together, this series of N-alkylcarbamates shows a FAAH-dependent inhibition of cellular AEA uptake, which can be strongly potentiated using specific head group modifications. These findings provide a rational basis for the development of hyperpotent AEA uptake inhibitors mediated by ultrapotent FAAH inhibition.     

Anti-atherogenic effects of statins: Impact on angiopoietin-2 release from endothelial cells

Beyond lipid lowering, statins are supposed to exert pleiotropic effects positively influencing the progression of atherosclerotic lesions. The development of such lesions is associated with increased release of angiopoietin-2 (Ang-2), an endothelial cell-specific protein growth factor stored in Weibel-Palade bodies (WPBs). The aim of our study was to examine whether statin pretreatment influences the release of Ang-2 from endothelial cells. Stimulation of HUVECs and HMVECs with PMA, thrombin or histamine resulted in significant release of Ang-2, as evidenced by ELISA. Pretreatment with simvastatin and mevastatin suppressed this release to basal level, while pravastatin had no effect. Simvastatin itself increased nitric oxide (NO, EC number 1.14.13.39) synthesis, measured by Griess reaction. Combining the statin pretreatment with the eNOS inhibitor L-NNA as well as bypassing the HMG-CoA reductase (EC number: 1.1.1.34) by adding mevalonic acid or geranyl pyrophosphate restored the exocytotic effect of PMA. Immunofluorescence microscopy showed that depletion of WPBs upon PMA stimulation ceased after pretreatment with simvastatin. This study demonstrates a potent suppressive effect of statins on the release of Ang-2 from endothelial cells. Regarding its harmful effects in the development of atherosclerotic lesions, our data provide further insight into the mechanisms of the anti-atherogenic potential of statins.     

Inhibition of STIM1 phosphorylation underlies resveratrol-induced inhibition of store-operated calcium entry

Resveratrol, a natural phytoalexin that shows health-promoting benefits, is an inhibitor of store-operated calcium entry (SOCE). Knowledge of the molecular mechanism underlying this inhibition is required for the proper design of therapies that include resveratrol or related stilbenoids, but remains largely unknown. To unravel this mechanism, using HEK293 cells as a model, we found that resveratrol inhibited the ERK1/2 activation triggered by Ca²⁺ store depletion. As a consequence, resveratrol inhibited STIM1 phosphorylation at residues Ser575, Ser608, and Ser621. Because this phosphorylation regulates the dissociation of STIM1 from the microtubule plus-end binding protein EB1 under store depletion conditions, resveratrol inhibited STIM1-EB1 dissociation. This inhibition had downstream effects such as inhibition of STIM1 multimerization in response to store depletion, and a significant impairment in the binding of STIM1 to ORAI1. Although additional targets for resveratrol in the molecular mechanism that governs SOCE cannot be discarded, the present results demonstrate that ERK1/2 pathway is a major target for resveratrol, and that the impairment of its activation produces a significant inhibition of SOCE.     

Influence of borneol and muscone on geniposide transport through MDCK and MDCK-MDR1 cells as blood–brain barrier in vitro model

The objective of this study was (1) to characterize geniposide transport through MDCK and MDCK-MDR1 cell lines to confirm its transport mechanism and (2) to evaluate the effect of borneol and muscone as enhancers of geniposide transport in the BBB models so as to explore the enhancement mechanism. Transport studies of geniposide were performed in both directions, from apical to basolateral and from basolateral to apical sides. Drug concentrations were analyzed by HPLC. Geniposide showed relatively poor absorption in MDCK and MDCK-MDR1 cells, apparent permeability coefficients ranging from 0.323 × 10⁻⁶ to 0.422 × 10⁻⁶ cm/s. The in vitro experiments showed that geniposide transport in both directions was not concentration dependent and saturable, indicating purely passive diffusion. The efflux ratio of geniposide was less than 2 in the two cell models, which suggested that geniposide was not P-gp substrates. Geniposide transport in both directions significantly increased when co-administrated with increasing concentrations of borneol and muscone. Actin staining results indicated that borneol and muscone increased geniposide transport in the BBB models may attribute to disassembly effect on tight junction integrity.     

The spirocyclopiperazinium salt compound LXM-15 alleviates chronic inflammatory and neuropathic pain in mice and rats

In the present study, we aimed to evaluate the effect of the spirocyclopiperazinium salt compound LXM-15 on chronic inflammatory pain and chronic neuropathic pain induced by complete Freund’s adjuvant (CFA) or chronic constriction injury (CCI) in mice and rats. The results showed that administration with LXM-15 significantly reduced paw edema and ankle swelling and increased the mechanical withdrawal threshold and paw withdrawal latency after CFA injection in mice. LXM-15 significantly alleviated the mechanical allodynia and thermal hyperalgesia in CCI rats. The effect was abolished by pretreatment with hexamethonium (a peripheral nAChR antagonist) or methyllycaconitine citrate (an α7 nAChR antagonist). Furthermore, LXM-15 significantly inhibited the phosphorylation of JAK2 and STAT3, and suppressed the expressions of TNF-α and c-fos in dorsal root ganglia of CCI rats. Collectively, we reported that LXM-15 relieved chronic inflammatory pain in CFA mice and chronic neuropathic pain in CCI rats. The underlying mechanism might be related to the activation of peripheral α7 nicotinic receptor, further inhibiting JAK2/STAT3 signaling pathway, and eventually suppressing the expressions of TNF-α and c-fos.