Monocyte NLRP3 inflammasome and interleukin-1ß activation modulated by alpha-1 antitrypsin therapy in deficient individuals.

INTRODUCTION: Altered complement component 3 (C3) activation in patients with alpha-1 antitrypsin (AAT) deficiency (AATD) has been reported. To understand the potential impact on course of inflammation, the aim of this study was to investigate whether C3d, a cleavage-product of C3, triggers interleukin (IL)-1ß secretion via activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. The objective was to explore the effect of AAT augmentation therapy in patients with AATD on the C3d/complement receptor 3 (CR3) signalling axis of monocytes and on circulating pro-inflammatory markers. METHODS: Inflammatory mediators were detected in blood from patients with AATD (n=28) and patients with AATD receiving augmentation therapy (n=19). Inflammasome activation and IL-1ß secretion were measured in monocytes of patients with AATD, and following C3d stimulation in the presence or absence of CR3 or NLRP3 inhibitors. RESULTS: C3d acting via CR3 induces NLRP3 and pro-IL-1ß production, and through induction of endoplasmic reticulum (ER) stress and calcium flux, triggers caspase-1 activation and IL-1ß secretion. Treatment of individuals with AATD with AAT therapy results in decreased plasma levels of C3d (3.0±1.2 µg/mL vs 1.3±0.5 µg/mL respectively, p<0.0001) and IL-1ß (115.4±30 pg/mL vs 73.3±20 pg/mL, respectively, p<0.0001), with a 2.0-fold decrease in monocyte NLRP3 protein expression (p=0.0303), despite continued ER stress activation. DISCUSSION: These results provide strong insight into the mechanism of complement-driven inflammation associated with AATD. Although the described variance in C3d and NLRP3 activation decreased post AAT augmentation therapy, results demonstrate persistent C3d and monocyte ER stress, with implications for new therapeutics and clinical practice.

Evaluating Novel Protein Phosphatase 2A Activators as Therapeutics for Emphysema.

The activity of PP2A (protein phosphatase 2A), a serine-threonine phosphatase, is reduced by chronic cigarette smoke (SM) exposure and α-1 antitrypsin (AAT) deficiency, and chemical activation of PP2A reduces the loss of lung function in SM-exposed mice. However, the previously studied PP2A-activator tricyclic sulfonamide compound DBK-1154 has low stability to oxidative metabolism, resulting in fast clearance and low systemic exposure. Here we compare the utility of a new more stable PP2A activator, ATUX-792, versus DBK-1154 for the treatment of SM-induced emphysema. ATUX-792 was also tested in human bronchial epithelial cells and a mouse model of AAT deficiency, Serpina1a-e-knockout mice. Human bronchial epithelial cells were treated with ATUX-792 or DBK-1154, and cell viability, PP2A activity, and MAP (mitogen-activated protein) kinase phosphorylation status were examined. Wild-type mice received vehicle, DBK-1154, or ATUX-792 orally in the last 2 months of 4 months of SM exposure, and 8-month-old Serpina1a-e-knockout mice received ATUX-792 daily for 4 months. Forced oscillation and expiratory measurements and histology analysis were performed. Treatment with ATUX-792 or DBK-1154 resulted in PP2A activation, reduced MAP kinase phosphorylation, immune cell infiltration, reduced airspace enlargements, and preserved lung function. Using protein arrays and multiplex assays, PP2A activation was observed to reduce AAT-deficient and SM-induced release of CXCL5, CCL17, and CXCL16 into the airways, which coincided with reduced neutrophil lung infiltration. Our study indicates that suppression of the PP2A activity in two models of emphysema could be restored by next-generation PP2A activators to impact lung function.

Ceramide contributes to pathogenesis and may be targeted for therapy in VCP inclusion body myopathy.

Knock-in homozygote VCPR155H/R155H mutant mice are a lethal model of valosin-containing protein (VCP)-associated inclusion body myopathy associated with Paget disease of bone, frontotemporal dementia and amyotrophic lateral sclerosis. Ceramide (d18:1/16:0) levels are elevated in skeletal muscle of the mutant mice, compared to wild-type controls. Moreover, exposure to a lipid-enriched diet reverses lethality, improves myopathy and normalizes ceramide levels in these mutant mice, suggesting that dysfunctions in lipid-derived signaling are critical to disease pathogenesis. Here, we investigated the potential role of ceramide in VCP disease using pharmacological agents that manipulate the ceramide levels in myoblast cultures from VCP mutant mice and VCP patients. Myoblasts from wild-type, VCPR155H/+ and VCPR155H/R155H mice, as well as patient-induced pluripotent stem cells (iPSCs), were treated with an inhibitor of ceramide degradation to increase ceramide via acid ceramidase (ARN082) for proof of principle. Three chemically distinct inhibitors of ceramide biosynthesis via serine palmitoyl-CoA transferase (L-cycloserine, myriocin or ARN14494) were used as a therapeutic strategy to reduce ceramide in myoblasts. Acid ceramidase inhibitor, ARN082, elevated cellular ceramide levels and concomitantly enhanced pathology. Conversely, inhibitors of ceramide biosynthesis L-cycloserine, myriocin and ARN14494 reduced ceramide production. The results point to ceramide-mediated signaling as a key contributor to pathogenesis in VCP disease and suggest that manipulating this pathway by blocking ceramide biosynthesis might exert beneficial effects in patients with this condition. The ceramide pathway appears to be critical in VCP pathogenesis, and small-molecule inhibitors of ceramide biosynthesis might provide therapeutic benefits in VCP and related neurodegenerative diseases.

Protein Phosphatase 2A as a Therapeutic Target in Pulmonary Diseases.

New disease targets and medicinal chemistry approaches are urgently needed to develop novel therapeutic strategies for treating pulmonary diseases. Emerging evidence suggests that reduced activity of protein phosphatase 2A (PP2A), a complex heterotrimeric enzyme that regulates dephosphorylation of serine and threonine residues from many proteins, is observed in multiple pulmonary diseases, including lung cancer, smoke-induced chronic obstructive pulmonary disease, alpha-1 antitrypsin deficiency, asthma, and idiopathic pulmonary fibrosis. Loss of PP2A responses is linked to many mechanisms associated with disease progressions, such as senescence, proliferation, inflammation, corticosteroid resistance, enhanced protease responses, and mRNA stability. Therefore, chemical restoration of PP2A may represent a novel treatment for these diseases. This review outlines the potential impact of reduced PP2A activity in pulmonary diseases, endogenous and exogenous inhibitors of PP2A, details the possible PP2A-dependent mechanisms observed in these conditions, and outlines potential therapeutic strategies for treatment. Substantial medicinal chemistry efforts are underway to develop therapeutics targeting PP2A activity. The development of specific activators of PP2A that selectively target PP2A holoenzymes could improve our understanding of the function of PP2A in pulmonary diseases. This may lead to the development of therapeutics for restoring normal PP2A responses within the lung.

Human Asymptomatic Epitopes Identified from the Herpes Simplex Virus Tegument Protein VP13/14 (UL47) Preferentially Recall Polyfunctional Effector Memory CD44high CD62Llow CD8+ TEM Cells and Protect Humanized HLA-A*02:01 Transgenic Mice against Ocular Herpesvirus Infection.

Herpes simplex virus 1 (HSV-1) infection is widespread among humans. The HSV-1 virion protein 13/14 (VP13/14), also known as UL47, is a tegument antigen targeted by CD8+ T cells from HSV-seropositive individuals. However, whether VP13/14-specific CD8+ T cells play a role in the natural protection seen in asymptomatic (ASYMP) individuals (individuals who have never had a clinical herpetic disease) has not been elucidated. Using predictive computer-assisted algorithms, we identified 10 potential HLA-A*02:01-restricted CD8+ T-cell epitopes from the 693-amino-acid sequence of the VP13/14 protein. Three out of 10 epitopes exhibited a high to moderate affinity of binding to soluble HLA-A*02:01 molecules. The phenotype and function of CD8+ T cells specific for each epitope were compared in HLA-A*02:01-positive ASYMP individuals and symptomatic (SYMP) individuals (individuals who have frequent clinical herpetic diseases) using determination of a combination of tetramer frequency and the levels of granzyme B, granzyme K, perforin, gamma interferon, tumor necrosis factor alpha, and interleukin-2 production and CD107a/b cytotoxic degranulation. High frequencies of multifunctional CD8+ T cells directed against three epitopes, VP13/14 from amino acids 286 to 294 (VP13/14286-294), VP13/14 from amino acids 504 to 512 (VP13/14504-512), and VP13/14 from amino acids 544 to 552 (VP13/14544-552), were detected in ASYMP individuals, while only low frequencies were detected in SYMP individuals. The three epitopes also predominantly recalled more CD45RAlow CD44high CCR7low CD62Llow CD8+ effector memory T cells (TEM cells) in ASYMP individuals than SYMP individuals. Moreover, immunization of HLA-A*02:01 transgenic mice with the three CD8+ TEM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8+ TEM cells associated with strong protective immunity against ocular herpesvirus infection and disease. Our findings outline the phenotypic and functional features of protective HSV-specific CD8+ T cells that should guide the development of a safe and effective T-cell-based herpes simplex vaccine. IMPORTANCE: Although most herpes simplex virus 1 (HSV-1)-infected individuals shed the virus in their body fluids following reactivation from latently infected sensory ganglia, the majority never develop a recurrent herpetic disease and remain asymptomatic (ASYMP). In contrast, small proportions of individuals are symptomatic (SYMP) and develop frequent bouts of recurrent disease. The present study demonstrates that naturally protected ASYMP individuals have a higher frequency of effector memory CD8+ T cells (CD8+ TEM cells) specific to three epitopes derived from the HSV-1 tegument protein VP13/14 (VP13/14286-294,VP13/14504-512, and VP13/14544-552) than SYMP patients. Moreover, immunization of humanized HLA-A*02:01 transgenic mice with the three CD8+ TEM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8+ T cells associated with strong protective immunity against ocular herpesvirus infection and disease. The findings support the emerging concept of the development of a safe and effective asymptomatic herpes simplex vaccine that is selectively based on CD8+ T-cell epitopes from ASYMP individuals.

Severe Hyponatremia (96 mmol/L) Secondary to Primary Polydipsia and Pneumonia.

A 63-year-old man who presented to the hospital with altered mental status and decreased responsiveness was found to have severe symptomatic hyponatremia with a sodium level of 96 mmol/L and pneumonia. The patient was admitted to the medical intensive care unit for septic shock and acute severe hyponatremia. He was intubated for airway protection, and treated with 3% hypertonic saline bolus and antibiotics. After four days, sodium levels were corrected to 128 mmol/L, and the patient was extubated and downgraded to the medical floor. This case demonstrates one of the lowest recorded sodium lab values ever and the patient was successfully treated and discharged home with appropriate outpatient appointments.

Low-Power Long-Duration Versus High-Power Short-Duration Radiofrequency Ablation of the Atrioventricular Node.

BACKGROUND: Atrioventricular node (AVN) radiofrequency (RF) ablation is a highly effective treatment of atrial tachyarrhythmias that are resistant to other management modalities. To date, there is limited research that compares the properties of different RF ablation catheters. The current study aims to compare the effectiveness of several types of RF catheters in AVN ablation. METHODS: 66 patients, with a mean age of 73.27 years, underwent AVN RF ablation. The catheters used were categorized as: un-irrigated (UI), externally-irrigated (EI), and contact force-sensing with 10-20 grams of force. EI catheters were divided into two different settings: low-power long-duration (LPLD) (30W, 45°C, and 60 sec) and high-power short-duration (HPSD) (50W, 43°C, and 12 sec). We compared the success rate of the different RF catheters using logistic regression and lesion times using linear regression. RESULTS: The distribution of the types of catheters used is: UI in 48%, LPLD in 16%, and HPSD in 36% of patients. All ablation procedures were successful, with no immediate post-procedure complications. HPSD had a significantly shorter lesion time than UI catheters by 403.42 sec [-631.67, -175.17]. CONCLUSION: UI catheters, LPLD, and HPSD were equally safe and effective in ablation procedures. The HPSD catheter had a significantly shorter lesion time and, thus, overall decreased procedure time.

Sinus Node Dysfunction Triggered by Tonsillar Abscess: Effects of Vagal Nerve Compression.

BACKGROUND Compression of the vagus nerve by a pharyngeal mass is a well-documented condition that can result in sinus node dysfunction (SND). However, there is scarce literature on extrinsic vagal nerve compression from a tonsillar abscess. CASE REPORT A 59-year-old woman with a history of asthma and chronic throat discomfort presented to the Emergency Department with bradycardia, palpitations, and voice changes. Following a shellfish allergy hospitalization, an otolaryngology evaluation revealed an enlarged right tonsil, recommending tonsillectomy, but scheduling challenges persisted. The patient reported mild throat pain, dysphagia, hoarseness, rhinorrhea, and exertional dyspnea and was admitted for the evaluation of peritonsillar mass. She was found to be bradycardic with a heart rate of 47, with an electrocardiogram revealing SND. Albuterol and ipratropium nebulizers, as well as dexamethasone and pantoprazole, were initiated. With this treatment, the patient symptomatically improved with a new heart rate of 68. She was discharged with outpatient appointments, but was unfortunately lost to follow-up. CONCLUSIONS This case reveals sinus node dysfunction resulting from extrinsic vagal nerve compression by a tonsillar abscess. Pressure on the vagus nerve can trigger bradycardia and low blood pressure, possibly due to compensatory overfiring of afferent vagal nerve signals from local mass effect. Early recognition and antibiotic treatment are essential to prevent cardiac complications. Clinicians must remain vigilant for such extrinsic causes, particularly in patients with chronic sore throat and cardiac symptoms. Further research and case reports are needed to deepen our understanding of this rare yet significant association.

Skeletal muscle effects of antisense oligonucleotides targeting glycogen synthase 1 in a mouse model of Pompe disease.

Pompe disease (PD) is a progressive myopathy caused by the aberrant accumulation of glycogen in skeletal and cardiac muscle resulting from the deficiency of the enzyme acid alpha-glucosidase (GAA). Administration of recombinant human GAA as enzyme replacement therapy (ERT) works well in alleviating the cardiac manifestations of PD but loses sustained benefit in ameliorating the skeletal muscle pathology. The limited efficacy of ERT in skeletal muscle is partially attributable to its inability to curb the accumulation of new glycogen produced by the muscle enzyme glycogen synthase 1 (GYS1). Substrate reduction therapies aimed at knocking down GYS1 expression represent a promising avenue to improve Pompe myopathy. However, finding specific inhibitors for GYS1 is challenging given the presence of the highly homologous GYS2 in the liver. Antisense oligonucleotides (ASOs) are chemically modified oligomers that hybridize to their complementary target RNA to induce their degradation with exquisite specificity. In the present study, we show that ASO-mediated Gys1 knockdown in the Gaa -/- mouse model of PD led to a robust reduction in glycogen accumulation in skeletal and cardiac muscle. In addition, combining Gys1 ASO with ERT further reduced glycogen content in muscle, eliminated autophagic buildup and lysosomal dysfunction, and improved motor function in Gaa -/- mice. Our results provide a strong foundation for further validation of the use of Gys1 ASO, alone or in combination with ERT, as a therapy for PD. We propose that early administration of Gys1 ASO in combination with ERT may be the key to preventative treatment options in PD.

Small-Molecule Activation of Protein Phosphatase 2A Counters Bleomycin-Induced Fibrosis in Mice.

The activity of protein phosphatase 2A (PP2A), a serine-threonine phosphatase, is reduced in the lung fibroblasts of idiopathic pulmonary fibrosis (IPF) patients. The objective of this study was to determine whether the reactivation of PP2A could reduce fibrosis and preserve the pulmonary function in a bleomycin (BLM) mouse model. Here, we present a new class of direct small-molecule PP2A activators, diarylmethyl-pyran-sulfonamide, exemplified by ATUX-1215. ATUX-1215 has improved metabolic stability and bioavailability compared to our previously described PP2A activators. Primary human lung fibroblasts were exposed to ATUX-1215 and an older generation PP2A activator in combination with TGFß. ATUX-1215 treatment enhanced the PP2A activity, reduced the phosphorylation of ERK and JNK, and reduced the TGFß-induced expression of ACTA2, FN1, COL1A1, and COL3A1. C57BL/6J mice were administered 5 mg/kg ATUX-1215 daily following intratracheal instillation of BLM. Three weeks later, forced oscillation and expiratory measurements were performed using the Scireq Flexivent System. ATUX-1215 prevented BLM-induced lung physiology changes, including the preservation of normal PV loop, compliance, tissue elastance, and forced vital capacity. PP2A activity was enhanced with ATUX-1215 and reduced collagen deposition within the lungs. ATUX-1215 also prevented the BLM induction of Acta2, Ccn2, and Fn1 gene expression. Treatment with ATUX-1215 reduced the phosphorylation of ERK, p38, JNK, and Akt and the secretion of IL-12p70, GM-CSF, and IL1α in BLM-treated animals. Delayed treatment with ATUX-1215 was also observed to slow the progression of lung fibrosis. In conclusion, our study indicates that the decrease in PP2A activity, which occurs in fibroblasts from the lungs of IPF subjects, could be restored with ATUX-1215 administration as an antifibrotic agent.

Steps in the bacterial flagellar motor.

The bacterial flagellar motor is a highly efficient rotary machine used by many bacteria to propel themselves. It has recently been shown that at low speeds its rotation proceeds in steps. Here we propose a simple physical model, based on the storage of energy in protein springs, that accounts for this stepping behavior as a random walk in a tilted corrugated potential that combines torque and contact forces. We argue that the absolute angular position of the rotor is crucial for understanding step properties and show this hypothesis to be consistent with the available data, in particular the observation that backward steps are smaller on average than forward steps. We also predict a sublinear speed versus torque relationship for fixed load at low torque, and a peak in rotor diffusion as a function of torque. Our model provides a comprehensive framework for understanding and analyzing stepping behavior in the bacterial flagellar motor and proposes novel, testable predictions. More broadly, the storage of energy in protein springs by the flagellar motor may provide useful general insights into the design of highly efficient molecular machines.

Modeling torque versus speed, shot noise, and rotational diffusion of the bacterial flagellar motor.

We present a minimal physical model for the flagellar motor that enables bacteria to swim. Our model explains the experimentally measured torque-speed relationship of the proton-driven E. coli motor at various pH and temperature conditions. In particular, the dramatic drop of torque at high rotation speeds (the "knee") is shown to arise from saturation of the proton flux. Moreover, we show that shot noise in the proton current dominates the diffusion of motor rotation at low loads. This suggests a new way to probe the discreteness of the energy source, analogous to measurements of charge quantization in superconducting tunnel junctions.

New family with HSPB8-associated autosomal dominant rimmed vacuolar myopathy.

OBJECTIVE: We clinically and molecularly characterize a new family with autosomal dominant rimmed vacuolar myopathy (RVM) caused by mutations in the HSPB8 gene. METHODS: We performed whole-exome and whole-genome sequencing in the family. Western blot and immunocytochemistry were used to analyze 3 patient fibroblasts, and findings were compared with their age- and sex-matched controls. RESULTS: Affected patients have distal and proximal myopathy, with muscle biopsy showing rimmed vacuoles, muscle fiber atrophy, and endomysial fibrosis typical of RVM. Muscle MRI showed severe relatively symmetric multifocal fatty degenerative changes of the lower extremities. We identified a duplication of C at position 515 of the HSPB8 gene (c.515dupC) by whole-genome sequencing, which caused a frameshift with a predicted alternate stop codon p.P173SFS*43 in all affected individuals, resulting in an elongated protein product. Western blot and immunocytochemistry studies revealed reduced expression of heat shock protein beta 8 in patient fibroblasts compared with control fibroblasts, in addition to disrupted autophagy pathology. CONCLUSIONS: We report a novel family with autosomal dominant RVM caused by the c.515dupC mutation of the HSPB8 gene, causing a translational frameshift that results in an elongated protein. Understanding the mechanism for the RVM pathology caused by mutated chaperone will permit novel targeted strategies to alter the natural history progression. As next-generation sequencing becomes more available, additional myopathic families will be identified with HSPB8 mutations.

Single molecule correlation spectroscopy in continuous flow mixers with zero-mode waveguides.

Zero-Mode Waveguides were first introduced for Fluorescence Correlation Spectroscopy at micromolar dye concentrations. We show that combining zero-mode waveguides with fluorescence correlation spectroscopy in a continuous flow mixer avoids the compression of the FCS signal due to fluid transport at channel velocities up to approximately 17 mm/s. We derive an analytic scaling relationship [equation: see text] converting this flow velocity insensitivity to improved kinetic rate certainty in time-resolved mixing experiments. Thus zero-mode waveguides make FCS suitable for direct kinetics measurements in rapid continuous flow.

Myogenic differentiation of VCP disease-induced pluripotent stem cells: A novel platform for drug discovery.

Valosin Containing Protein (VCP) disease is an autosomal dominant multisystem proteinopathy caused by mutations in the VCP gene, and is primarily associated with progressive muscle weakness, including atrophy of the pelvic and shoulder girdle muscles. Currently, no treatments are available and cardiac and respiratory failures can lead to mortality at an early age. VCP is an AAA ATPase multifunction complex protein and mutations in the VCP gene resulting in disrupted autophagic clearance. Due to the rarity of the disease, the myopathic nature of the disorder, ethical and practical considerations, VCP disease muscle biopsies are difficult to obtain. Thus, disease-specific human induced pluripotent stem cells (hiPSCs) now provide a valuable resource for the research owing to their renewable and pluripotent nature. In the present study, we report the differentiation and characterization of a VCP disease-specific hiPSCs into precursors expressing myogenic markers including desmin, myogenic factor 5 (MYF5), myosin and heavy chain 2 (MYH2). VCP disease phenotype is characterized by high expression of TAR DNA Binding Protein-43 (TDP-43), ubiquitin (Ub), Light Chain 3-I/II protein (LC3-I/II), and p62/SQSTM1 (p62) protein indicating disruption of the autophagy cascade. Treatment of hiPSC precursors with autophagy stimulators Rapamycin, Perifosine, or AT101 showed reduction in VCP pathology markers TDP-43, LC3-I/II and p62/SQSTM1. Conversely, autophagy inhibitors chloroquine had no beneficial effect, and Spautin-1 or MHY1485 had modest effects. Our results illustrate that hiPSC technology provide a useful platform for a rapid drug discovery and hence constitutes a bridge between clinical and bench research in VCP and related diseases.

Chemical Vapor Deposition of an Organic Magnet, Vanadium Tetracyanoethylene.

Recent progress in the field of organic materials has yielded devices such as organic light emitting diodes (OLEDs) which have advantages not found in traditional materials, including low cost and mechanical flexibility. In a similar vein, it would be advantageous to expand the use of organics into high frequency electronics and spin-based electronics. This work presents a synthetic process for the growth of thin films of the room temperature organic ferrimagnet, vanadium tetracyanoethylene (V[TCNE]x, x~2) by low temperature chemical vapor deposition (CVD). The thin film is grown at <60 °C, and can accommodate a wide variety of substrates including, but not limited to, silicon, glass, Teflon and flexible substrates. The conformal deposition is conducive to pre-patterned and three-dimensional structures as well. Additionally this technique can yield films with thicknesses ranging from 30 nm to several microns. Recent progress in optimization of film growth creates a film whose qualities, such as higher Curie temperature (600 K), improved magnetic homogeneity, and narrow ferromagnetic resonance line-width (1.5 G) show promise for a variety of applications in spintronics and microwave electronics.

Thin-film deposition of an organic magnet based on vanadium methyl tricyanoethylenecarboxylate.

The preparation and characterization of a new thin-film organic-based magnet V[MeTCEC]x (V = vanadium; MeTCEC = methyl tricaynoethylenecarboxylate) via low-temperature chemical vapor deposition (50 °C) is reported. These thin films exhibit room-temperature magnetic ordering and semiconducting behavior, demonstrating the ability of tuning their magnetic, and potentially spintronic, functionality via chemical modification of the organic ligand.

Icariside II potentiates paclitaxel-induced apoptosis in human melanoma A375 cells by inhibiting TLR4 signaling pathway.

Combination therapy of paclitaxel (taxol) with natural anti-tumor agents that are capable of inhibiting survival signals may provide a rational molecular basis for novel chemotherapeutic strategies. Our previous study showed that icariside II (IS), derived from Herba Epimedii, inhibited the proliferation of melanoma cells in vivo and in vitro through the regulation of apoptosis. In this report, the combination effects of paclitaxel and IS were investigated in human melanoma A375 cells. As compared to the treatment with paclitaxel alone, the co-administration of IS and paclitaxel resulted in an enhancement of apoptosis as revealed by WST-8 and PI assays. Meanwhile, Western blot analysis showed that the co-administration of IS and paclitaxel resulted in increases of cleaved caspase-3, one of the terminal pro-apoptotic proteins. In melanoma, IL-8 and VEGF are positively correlated with disease stage and a high probability of progression. We demonstrated that treatment of A375 cells with IS in combination with paclitaxel resulted in a significant decrease in the production of IL-8 and VEGF, compared with paclitaxel alone. Recent studies suggest that TLR4-MyD88-ERK signaling may be a novel target for reversing chemoresistance to paclitaxel. Our flow cytometry and Western blot data showed that paclitaxel activated TLR4-MyD88-ERK signaling and that IS treatment could effectively inhibit this paclitaxel-induced activation of TLR4-MyD88-ERK signaling. In conclusion, this study demonstrated for the first time that IS could potentiate paclitaxel-induced apoptosis in melanoma cells. These effects were mediated, at least in part, by inhibiting the activation of the TLR4 signal transduction pathways. These findings support further preclinical evaluation of IS as a new potential anti-tumor agent.