Targeting epidermal growth factor receptor in paclitaxel-resistant human breast and lung cancer cells with upregulated glucose-6-phosphate dehydrogenase.

BACKGROUND: Chemoresistance is a major obstacle to the successful treatment of triple-negative breast cancer (TNBC) and non-small-cell lung cancer (NSCLC). Therapeutic strategies to overcome chemoresistance are necessary to improve the prognosis of patients with these cancers. METHODS: Paclitaxel-resistant TNBC and NSCLC sublines were generated through continuous paclitaxel treatment over 6 months. The mechanistic investigation was conducted using MTT assay, LC/MS-based metabolite analysis, flow cytometry, western blot analysis, real-time PCR and tumour xenograft experiments. RESULTS: Glucose-6-phosphate dehydrogenase (G6PD) expression along with an increase in 3-phosphoglycerates and ribulose-5-phosphate production was upregulated in paclitaxel-resistant cells. Blockade of G6PD decreased viability of paclitaxel-resistant cells in vitro and the growth of paclitaxel-resistant MDA/R xenograft tumours in vivo. Mechanistically, activation of the epidermal growth factor receptor (EGFR)/Akt pathway mediates G6PD expression and G6PD-induced cell survival. Blockade of the EGFR pathway inhibited G6PD expression and sensitised those paclitaxel-resistant cells to paclitaxel treatment in vitro and in vivo. Analysis of publicly available datasets revealed an association between G6PD and unfavourable clinical outcomes in patients with breast or lung cancer. CONCLUSIONS: EGFR signaling-mediated G6PD expression plays a pivotal role in paclitaxel resistance, highlighting the potential of targeting EGFR to overcome paclitaxel resistance in TNBC and NSCLC cells overexpressing G6PD.

Targeting the insulin-like growth factor receptor and Src signaling network for the treatment of non-small cell lung cancer.

BACKGROUND: Therapeutic interventions in the insulin-like growth factor receptor (IGF-1R) pathway were expected to provide clinical benefits; however, IGF-1R tyrosine kinase inhibitors (TKIs) have shown limited antitumor efficacy, and the mechanisms conveying resistance to these agents remain elusive. METHODS: The expression and activation of the IGF-1R and Src were assessed via the analysis of a publicly available dataset, as well as immunohistochemistry, Western blotting, RT-PCR, and in vitro kinase assays. The efficacy of IGF-1R TKIs alone or in combination with Src inhibitors was analyzed using MTT assays, colony formation assays, flow cytometric analysis, and xenograft tumor models. RESULTS: The co-activation of IGF-1R and Src was observed in multiple human NSCLC cell lines as well as in a tissue microarray (n = 353). The IGF-1R and Src proteins mutually phosphorylate on their autophosphorylation sites. In high-pSrc-expressing NSCLC cells, linsitinib treatment initially inactivated the IGF-1R pathway but led a Src-dependent reactivation of downstream effectors. In low-pSrc-expressing NSCLC cells, linsitinib treatment decreased the turnover of the IGF-1R and Src proteins, ultimately amplifying the reciprocal co-activation of IGF-1R and Src. Co-targeting IGF-1R and Src significantly suppressed the proliferation and tumor growth of both high-pSrc-expressing and low-pSrc-expressing NSCLC cells in vitro and in vivo and the growth of patient-derived tissues in vivo. CONCLUSIONS: Reciprocal activation between Src and IGF-1R occurs in NSCLC. Src causes IGF-1R TKI resistance by acting as a key downstream modulator of the cross-talk between multiple membrane receptors. Targeting Src is a clinically applicable strategy to overcome resistance to IGF-1R TKIs.

The novel TAK1 inhibitor handelin inhibits NF-κB and AP-1 activity to alleviate elastase-induced emphysema in mice.

AIMS: Emphysema, one of the two major components of chronic obstructive pulmonary disease (COPD), is driven by aberrant inflammatory responses and associated with irreversible lung parenchymal destruction. As effective therapy for preventing or treating COPD/emphysema is yet unavailable, development of molecular targets and therapeutic agents for COPD/emphysema is required. MAIN METHODS AND KEY FINDINGS: We identified handelin-a guaianolide dimer of sesquiterpene lactones- from a chemical library of 431 natural products as it exhibited potent inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) and reactive oxygen species (ROS) production, LPS-induced activation of nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK)/AP-1, and expression of proinflammatory mediators in macrophage cells. In silico docking and biochemical studies enabled the identification of the ATP-binding pocket of transforming growth factor beta-activated kinase 1 (TAK1), a kinase upstream of NF-κB and MAPK/AP-1 pathways, as a molecular target for handelin. Moreover, oral administration of handelin (10 mg/kg) suppressed elastase-induced development of emphysematous phenotypes, including lung function disturbance, airspace enlargement, and increases in the level of neutrophils and CD8+ T cells in lung tissues, without overt toxicity. Consistent with in vitro results, analyses of lung tissues revealed that treatment with handelin suppressed elastase-induced NF-κB and AP-1 activation in the lungs, followed by downregulation of their targets including interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and matrix metalloproteinase 9 (MMP9). SIGNIFICANCE: These findings suggest that handelin, as a TAK1 inhibitor, effectively prevents development of emphysema in an elastase-induced mouse model by inhibiting a proinflammatory mediators mediated by NF-κB and AP-1.

Gliotoxin enhances radiotherapy via inhibition of radiation-induced GADD45a, p38, and NFkappaB activation.

The purpose of the study was to elucidate the mechanism underlying the enhancement of radiosensitivity to 60Co gamma-irradiation in human hepatoma cell line HepG2 pretreated with gliotoxin. Enhancement of radiotherapy by gliotoxin was investigated in vitro with human hepatoma HepG2 cell line. Apoptosis related proteins were evaluated by Western blotting. Annexin V/PI and reactive oxygen species (ROS) were quantified by Flow Cytometric (FACS) analysis. Gliotoxin (200 ng/ml) combined with radiation (4 Gy) treated cells induced apoptosis. Cells treated with gliotoxin (200 ng/ml) prior to irradiation at 4 Gy induced the expression of bax and nitric oxide (NO). The gliotoxin-irradiated cells also increased caspase-3 activation and ROS. Gadd45a, p38, and nuclear factor kappa B (NFkappaB) activated in irradiated cells was inhibited by Gliotoxin. Specific inhibitors of p38 kinase, SB203580, significantly inhibited NFkappaB activation and increased the cytotoxicity effect in cells exposed to gliotoxin combined with irradiation. However, SB203580 did not suppress the activation of Gadd45a in irradiated cells. Gliotoxin inhibited anti-apoptotic signal pathway involving the activation of Gadd45a-p38-NFkappaB mediated survival pathway that prevent radiation-induced cell death. Therefore, gliotoxin, blocking inflammation pathway and enhancing irradiation-induced apoptosis, is a promising agent to increase the radiotherapy of tumor cells.

Smoking-associated lung cancer prevention by blockade of the beta-adrenergic receptor-mediated insulin-like growth factor receptor activation.

Activation of receptor tyrosine kinases (RTKs) is associated with carcinogenesis, but its contribution to smoking-associated lung carcinogenesis is poorly understood. Here we show that a tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced insulin-like growth factor 1 receptor (IGF-1R) activation via ß-adrenergic receptor (ß-AR) is crucial for smoking-associated lung carcinogenesis. Treatment with NNK stimulated the IGF-1R signaling pathway in a time- and dose-dependent manner, which was suppressed by pharmacological or genomic blockade of ß-AR and the downstream signaling including a Gßγ subunit of ß-AR and phospholipase C (PLC). Consistently, ß-AR agonists led to increased IGF-1R phosphorylation. The increase in IGF2 transcription via ß-AR, signal transducer and activator of transcription 3 (STAT3), and nuclear factor-kappa B (NF-κB) was associated with NNK-induced IGF-1R activation. Finally, treatment with ß-AR antagonists suppressed the acquisition of transformed phenotypes in lung epithelial cells and lung tumor formation in mice. These results suggest that blocking ß-AR-mediated IGF-1R activation can be an effective strategy for lung cancer prevention in smokers.

The tobacco-specific carcinogen-operated calcium channel promotes lung tumorigenesis via IGF2 exocytosis in lung epithelial cells.

Nicotinic acetylcholine receptors (nAChRs) binding to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces Ca2+ signalling, a mechanism that is implicated in various human cancers. In this study, we investigated the role of NNK-mediated Ca2+ signalling in lung cancer formation. We show significant overexpression of insulin-like growth factors (IGFs) in association with IGF-1R activation in human preneoplastic lung lesions in smokers. NNK induces voltage-dependent calcium channel (VDCC)-intervened calcium influx in airway epithelial cells, resulting in a rapid IGF2 secretion via the regulated pathway and thus IGF-1R activation. Silencing nAChR, α1 subunit of L-type VDCC, or various vesicular trafficking curators, including synaptotagmins and Rabs, or blockade of nAChR/VDCC-mediated Ca2+ influx significantly suppresses NNK-induced IGF2 exocytosis, transformation and tumorigenesis of lung epithelial cells. Publicly available database reveals inverse correlation between use of calcium channel blockers and lung cancer diagnosis. Our data indicate that NNK disrupts the regulated pathway of IGF2 exocytosis and promotes lung tumorigenesis.

Vitamin C Stimulates Epidermal Ceramide Production by Regulating Its Metabolic Enzymes.

Ceramide is the most abundant lipid in the epidermis and plays a critical role in maintaining epidermal barrier function. Overall ceramide content in keratinocyte increases in parallel with differentiation, which is initiated by supplementation of calcium and/or vitamin C. However, the role of metabolic enzymes responsible for ceramide generation in response to vitamin C is still unclear. Here, we investigated whether vitamin C alters epidermal ceramide content by regulating the expression and/or activity of its metabolic enzymes. When human keratinocytes were grown in 1.2 mM calcium with vitamin C (50 µg/ml) for 11 days, bulk ceramide content significantly increased in conjunction with terminal differentiation of keratinocytes as compared to vehicle controls (1.2 mM calcium alone). Synthesis of the ceramide fractions was enhanced by increased de novo ceramide synthesis pathway via serine palmitoyltransferase and ceramide synthase activations. Moreover, sphingosine-1-phosphate (S1P) hydrolysis pathway by action of S1P phosphatase was also stimulated by vitamin C supplementation, contributing, in part, to enhanced ceramide production. However, activity of sphingomyelinase, a hydrolase enzyme that converts sphingomyelin to ceramide, remained unaltered. Taken together, we demonstrate that vitamin C stimulates ceramide production in keratinocytes by modulating ceramide metabolic-related enzymes, and as a result, could improve overall epidermal barrier function.

Novel C6-substituted 1,3,4-oxadiazinones as potential anti-cancer agents.

The insulin-like growth factor 1 receptor (IGF-1R) is a membrane receptor tyrosine kinase over-expressed in a number of tumors. However, combating resistance is one of the main challenges in the currently available IGF-1R inhibitor-based cancer therapies. Increased Src activation has been reported to confer resistance to anti-IGF-1R therapeutics in various tumor cells. An urgent unmet need for IGF-1R inhibitors is to suppress Src rephosphorylation induced by current anti-IGF-1R regimens. In efforts to develop effective anticancer agents targeting the IGF-1R signaling pathway, we explored 2-aryl-1,3,4-oxadiazin-5-ones as a novel scaffold that is structurally unrelated to current tyrosine kinase inhibitors (TKIs). The compound, LL-2003, exhibited promising antitumor effects in vitro and in vivo; it effectively suppressed IGF-1R and Src and induced apoptosis in various non-small cell lung cancer cells. Further optimizations for enhanced potency in cellular assays need to be followed, but our strategy to identify novel IGF-1R/Src inhibitors may open a new avenue to develop more efficient anticancer agents.

A novel antitumor activity of deguelin targeting the insulin-like growth factor (IGF) receptor pathway via up-regulation of IGF-binding protein-3 expression in breast cancer.

In this study, we investigated the antitumor effects of deguelin in several human breast cancer cells in vitro and in vivo. Deguelin inhibited cell viability and the anchorage-dependent and anchorage-independent colony formation of triple-negative (MDA-MB-231 and MDA-MB-468) and triple-positive (MCF-7) breast cancer cells, and it significantly reduced the growth of MCF-7 cell xenograft tumors. The induction of apoptosis, inhibition of insulin-like growth factor-1 receptor (IGF-1R) signaling activation, and up-regulation of IGF-binding protein-3 (IGFBP-3) expression may be associated with deguelin-mediated antitumor effects. Our findings suggest a potential therapeutic use for deguelin in patients with triple-negative breast cancer and for those with breast cancers who are sensitive to endocrine- and HER2-targeted therapies.

Comparison of sample preparation methods for the recovery of foodborne pathogens from fresh produce.

Sample preparation methods (pummeling, pulsifying, sonication, and shaking by hand) were compared for achieving maximum recovery of foodborne pathogens from iceberg lettuce, perilla leaves, cucumber, green pepper, and cherry tomato. Antimicrobial and dehydration effects also were examined to investigate causes of poor recovery of pathogens. Each produce type was inoculated with Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus at 6.0 log CFU/cm(2), and samples were prepared using the four methods. Bacterial populations recovered from the five types of produce were significantly different (P < 0.05) according to sample preparation methods and produce type. The bacterial populations recovered from pummeled and pulsified samples were higher (P < 0.05) than those recovered from sonicated and hand-shaken samples, except for cherry tomato. The number of bacteria recovered from produce was reduced (P < 0.05) from that of the inoculum by 0.16 to 2.69 log CFU/cm(2). Although extracts of iceberg lettuce, perilla leaves, cucumber, and green pepper had no antimicrobial activity, the populations of E. coli O157:H7, Salmonella Typhimurium, B. cereus, and L. monocytogenes in cherry tomato extract were slightly reduced after these treatments (P < 0.05). The pathogen populations on perilla leaves and cherry tomatoes decreased by >2 log CFU/cm(2) after exposure to 40% relative humidity for 1 h. No reduction was observed when the five pathogens were exposed to 90% relative humidity. These data suggest that pummeling and pulsifying are optimal sample preparation methods for detection of microorganisms. Acidic produce such as cherry tomato should be treated with a method that does not cause sample breakdown so that acid stress on the bacteria can be minimized. Dehydration stress also affects recovery of pathogens from produce.

The effects of magnesium on the course of atrial fibrillation and coagulation in patients with atrial fibrillation undergoing mitral valve annuloplasty.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Magnesium has been reported to be effective in reducing the incidence or prophylaxis of AF. Magnesium is also an essential constituent of many enzyme systems and plays a physiological role in coagulation regulation. The aim of the present study was to examine the effects of magnesium, whether magnesium infusion might decrease the incidence of AF and induce hypocoagulable state in patients with AF, who were undergoing mitral valve annuloplasty. METHODS: This prospective laboratory study was performed using blood from patients with AF undergoing mitral valve annuloplasty. The radial artery was punctured with a 20 gauge catheter and used for monitoring continuous arterial pressure and blood sampling. After anesthesia induction, 4 g of magnesium was mixed with 100 ml normal saline and infused for 5 minutes. Magnesium, calcium, activated clotting time (ACT) and thromboelastographic parameters were checked before and 60 minutes after the magnesium infusion. The electrocardiography changes after magnesium infusion were also checked before commencing cardiopulmonary bypass. RESULTS: After magnesium infusion, the serum level of magnesium increased significantly but serum calcium did not change significantly. ACT did not change significantly before or after magnesium infusion. The thromboelastographic parameters showed no significant changes before or after magnesium infusion. None of the patients converted to sinus rhythm from AF after the magnesium infusion. CONCLUSIONS: A magnesium infusion did not influence the course of AF and coagulation in patients during prebypass period with AF undergoing mitral valve annuloplasty.