The Substitution of Fifty Percent of Combustible Tobacco Smoke Exposure With Either Electronic Cigarettes or Heated tobacco Products Did Not Attenuate Acute Lung Injury in an Animal Model.

BACKGROUND: To reduce the harmful health effects of combustible cigarette smoke (CS), some (CS) users attempt to substitute CS with electronic cigarettes (ECIG) and/or heated tobacco products (HTP). In this animal study, we evaluated the acute effects of substituting CS consumption with ECIG or HTP thus mimicking the dual users' approach, on the lungs of a mouse model. METHODS: C57BL/6 mice were divided into Control, ECIG, HTP, CS, ECIG + CS, HTP + CS, and HTP + ECIG groups. Animals were exposed for 3 hours in AM and PM sessions to either air, CS, ECIG, or HTP for seven days. Lung injury was assessed by: wet to dry (W/D) ratio, albumin concentration in bronchoalveolar lavage fluid, expression of IL-1ß, IL-6, and TNF-α, histopathology examination, reactive oxygen species (ROS) production, and assessment of cellular apoptosis. RESULTS: W/D ratio was significantly increased in mice exposed to CS only. Albumin leak and expression of IL-1ß, IL-6, and TNF-a were elevated in CS, ECIG + CS, and HTP + CS. Histological examination revealed significant inflammatory cells infiltration, as well as collagen deposit in CS, ECIG + CS, HTP + CS. ROS production was significantly increased in CS, ECIG + CS, HTP + CS. Finally, cell death was also significantly increased in CS, ECIG + CS, and HTP + CS. CONCLUSION: In this animal model, substituting 50% of daily CS exposure by either ECIG or HTP exposure did not result in significant attenuation of acute lung injury.

Ubiquitin-Related Modifier 1 (URM-1) Modulates Cx43 in Breast Cancer Cell Lines.

Gap-junction-forming connexins are exquisitely regulated by post-translational modifications (PTMs). In particular, the PTM of connexin 43 (Cx43), a tumor suppressor protein, regulates its turnover and activity. Here, we investigated the interaction of Cx43 with the ubiquitin-related modifier 1 (URM-1) protein and its impact on tumor progression in two breast cancer cell lines, highly metastatic triple-negative MDA-MB-231 and luminal breast cancer MCF-7 cell lines. To evaluate the subsequent modulation of Cx43 levels, URM-1 was downregulated in these cells. The transcriptional levels of epithelial-to-mesenchymal transition (EMT) markers and the metastatic phenotype were assessed. We demonstrated that Cx43 co-localizes and interacts with URM-1, and URMylated Cx43 was accentuated upon cellular stress. The significant upregulation of small ubiquitin-like modifier-1 (SUMO-1) was also observed. In cells with downregulated URM-1, Cx43 expression significantly decreased, and SUMOylation by SUMO-1 was affected. The concomitant expression of EMT markers increased, leading to increased proliferation, migration, and invasion potential. Inversely, the upregulation of URM-1 increased Cx43 expression and reversed EMT-induced processes, underpinning a role for this PTM in the observed phenotypes. This study proposes that the URMylation of Cx43 in breast cancer cells regulates its tumor suppression properties and contributes to breast cancer cell malignancy.

Myeloperoxidase-Oxidized LDL Activates Human Aortic Endothelial Cells through the LOX-1 Scavenger Receptor.

Cardiovascular disease as a result of atherosclerosis is a leading cause of death worldwide. Atherosclerosis is primarily caused by the dysfunction of vascular endothelial cells and the subendothelial accumulation of oxidized forms of low-density lipoprotein (LDL). Early observations have linked oxidized LDL effects in atherogenesis to the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) scavenger receptor. It was shown that LOX-1 is upregulated by many inflammatory mediators and proatherogenic stimuli including cytokines, reactive oxygen species (ROS), hemodynamic blood flow, high blood sugar levels and, most importantly, modified forms of LDL. Oxidized LDL signaling pathways in atherosclerosis were first explored using LDL that is oxidized by copper (Cuox-LDL). In our study, we used a more physiologically relevant model of LDL oxidation and showed, for the first time, that myeloperoxidase oxidized LDL (Mox-LDL) may affect human aortic endothelial cell (HAEC) function through the LOX-1 scavenger receptor. We report that Mox-LDL increases the expression of its own LOX-1 receptor in HAECs, enhancing inflammation and simultaneously decreasing tubulogenesis in the cells. We hypothesize that Mox-LDL drives endothelial dysfunction (ED) through LOX-1 which provides an initial hint to the pathways that are initiated by Mox-LDL during ED and the progression of atherosclerosis.

EAPB0503, an Imidazoquinoxaline Derivative Modulates SENP3/ARF Mediated SUMOylation, and Induces NPM1c Degradation in NPM1 Mutant AML.

Nucleophosmin-1 (NPM1) is a pleiotropic protein involved in numerous cellular processes. NPM1 shuttles between the nucleus and the cytoplasm, but exhibits a predominant nucleolar localization, where its fate and functions are exquisitely controlled by dynamic post-translational modifications (PTM). Sentrin/SUMO Specific Peptidase 3 (SENP3) and ARF are two nucleolar proteins involved in NPM1 PTMs. SENP3 antagonizes ARF-mediated NPM1 SUMOylation, to promote ribosomal biogenesis. In Acute Myeloid Leukemia (AML), NPM1 is frequently mutated, and exhibits an aberrant cytoplasmic localization (NPM1c). NPM1c mutations define a separate AML entity with good prognosis in some AML patients, rendering NPM1c as a potential therapeutic target. SENP3-mediated NPM1 de-SUMOylation induces resistance to therapy in NPM1c AML. Here, we demonstrate that the imidazoquinoxaline EAPB0503 prolongs the survival and results in selective reduction in the leukemia burden of NPM1c AML xenograft mice. Indeed, EAPB0503 selectively downregulates HDM2 expression and activates the p53 pathway in NPM1c expressing cells, resulting in apoptosis. Importantly, we unraveled that NPM1c expressing cells exhibit low basal levels of SUMOylation paralleled with high SENP3 and low ARF basal levels. EAPB0503 reverted these molecular players by inducing NPM1c SUMOylation and ubiquitylation, leading to its proteasomal degradation. EAPB0503-induced NPM1c SUMOylation is concurrent with SENP3 downregulation and ARF upregulation in NPM1c expressing cells. Collectively, these results provide a strong rationale for testing therapies modulating NPM1c post-translational modifications in the management of NPM1c AML.

The Lebanese Red Algae Jania rubens: Promising Biomolecules against Colon Cancer Cells.

Colorectal cancer (CRC) is ranked the second most lethal type of tumor globally. Thus, developing novel anti-cancer therapeutics that are less aggressive and more potent is needed. Recently, natural bioactive molecules are gaining interest as complementary and supportive antineoplastic treatments due to their safety, effectiveness, and low cost. Jania rubens (J. rubens) is a red coral seaweed abundant in the Mediterranean and bears a significant pharmacological essence. Despite its therapeutic potential, the natural biomolecules extracted from this alga are poorly identified. In this study, the proximal analysis revealed high levels of total ash content (66%), 11.3% proteins, 14.5% carbohydrates, and only 4.5% lipids. The elemental identification showed magnesium and calcium were high among its macro minerals, (24 ± 0.5 mg/g) and (33 ± 0.5 mg/g), respectively. The Chlorophyll of J. rubens was dominated by other pigments with (0.82 ± 0.02 mg/g). A 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay identified effective antioxidant activity in various J. rubens extracts. More importantly, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium reduction and wound healing assays indicate that organic extracts from J. rubens significantly counteract the proliferation of colon cancer cell lines (HCT-116 and HT-29) and inhibit their migratory and metastatic properties in a dose and time-dependent manner. Overall, this study provides insight into the physicochemical properties of red seaweed, J. rubens, and identifies its significant antioxidant, cytotoxic, and anti-migratory potential on two colorectal cell lines, HCT-116 and HT-29.

Loss of interleukin-10 activates innate immunity to eradicate adult T-cell leukemia-initiating cells.

Adult T cell leukemia/lymphoma (ATL) is associated to chronic human T cell leukemia virus type 1 (HTLV-1) infection and carries a poor prognosis. Arsenic trioxide (AS) and interferon-alpha (IFNα) together selectively trigger Tax viral oncoprotein degradation and cure Tax-driven murine ATL. AS/IFNα/zidovudine treatment achieves a high response rate in patients with chronic ATL. Interleukin 10 (IL-10) is an immuno-suppressive cytokine whose expression is activated by Tax. Here we show that, in ATL, AS/IFNα-induced abrogation of leukemia initiating cell activity requires IL-10 expression shutoff. Loss of IL-10 secretion drives production of inflammatory cytokines by the microenvironment, followed by innate immunity-mediated clearance of Taxdriven leukemic cells. Accordingly, anti-IL-10 monoclonal antibodies significantly increased the efficiency of AS/IFNα therapy. These results emphasize the sequential targeting of malignant ATL cells and their immune microenvironment in leukemia initiating cell (LIC) eradication and provide a strong rational to test AS/IFNα/anti-IL10 combination in ATL.

A Pin1/PML/P53 axis activated by retinoic acid in NPM-1c acute myeloid leukemia.

Retinoic acid (RA) was proposed to increase survival of chemotherapy- treated patients with nucleophosmin-1 (NPM-1c)-mutated acute myeloid leukemia. We reported that, ex vivo, RA triggers NPM-1c degradation, P53 activation and growth arrest. PML organizes domains that control senescence or proteolysis. Here, we demonstrate that PML is required to initiate RA-driven NPM-1c degradation, P53 activation and cell death. Mechanistically, RA enhances PML basal expression through inhibition of activated Pin1, prior to NPM-1c degradation. Such PML induction drives P53 activation, favoring blast response to chemotherapy or arsenic in vivo. This RA/PML/P53 cascade could mechanistically explain RA-facilitated chemotherapy response in patients with NPM-1c mutated acute myeloid leukemia.

Dextran Sodium Sulfate-Induced Impairment of Protein Trafficking and Alterations in Membrane Composition in Intestinal Caco-2 Cell Line.

A key morphological feature of inflammatory bowel disease (IBD) is the loss of the barrier function of intestinal epithelial cells. The present study investigates endoplasmic reticulum (ER) stress in addition to alterations in protein and membrane trafficking in a dextran sulfate sodium (DSS)-induced IBD-like phenotype of intestinal Caco-2 cells in culture. DSS treatment significantly reduced the transepithelial electric resistance (TEER) and increased the epithelial permeability of Caco-2 cells, without affecting their viability. This was associated with an alteration in the expression levels of inflammatory factors in addition to an increase in the expression of the ER stress protein markers, namely immunoglobulin-binding protein (BiP), C/EBP homologous protein (CHOP), activation transcription factor 4 (ATF4), and X-box binding protein (XBP1). The DSS-induced ER-stress resulted in impaired intracellular trafficking and polarized sorting of sucrase-isomaltase (SI) and dipeptidyl peptidase-4 (DPPIV), which are normally sorted to the apical membrane via association with lipid rafts. The observed impaired sorting was caused by reduced cholesterol levels and subsequent distortion of the lipid rafts. The data presented confirm perturbation of ER homeostasis in DSS-treated Caco-2 cells, accompanied by impairment of membrane and protein trafficking resulting in altered membrane integrity, cellular polarity, and hence disrupted barrier function.

Gap Junctions and Wnt Signaling in the Mammary Gland: a Cross-Talk?

Connexins (Cxs), the building blocks of gap junctions (GJs), exhibit spatiotemporal patterns of expression and regulate the development and differentiation of the mammary gland, acting via channel-dependent and channel-independent mechanisms. Impaired Cx expression and localization are reported in breast cancer, suggesting a tumor suppressive role for Cxs. The signaling events that mediate the role of GJs in the development and tumorigenesis of the mammary gland remain poorly identified. The Wnt pathways, encompassing the canonical or the Wnt/ß-catenin pathway and the noncanonical ß-catenin-independent pathway, also play important roles in those processes. Indeed, aberrant Wnt signaling is associated with breast cancer. Despite the coincident roles of Cxs and Wnt pathways, the cross-talk in the breast tissue is poorly defined, although this is reported in a number of other tissues. Our previous studies revealed a channel-independent role for Cx43 in inducing differentiation or suppressing tumorigenesis of mammary epithelial cells by acting as a negative regulator of the Wnt/ß-catenin pathway. Here, we provide a brief overview of mammary gland development, with emphasis on the role of Cxs in development and tumorigenesis of this tissue. We also discuss the role of Wnt signaling in similar contexts, and review the literature illustrating interplay between Cxs and Wnt pathways.

Impaired Endothelium-Dependent Hyperpolarization Underlies Endothelial Dysfunction during Early Metabolic Challenge: Increased ROS Generation and Possible Interference with NO Function.

Endothelial dysfunction is a hallmark of diabetic vasculopathies. Although hyperglycemia is believed to be the culprit causing endothelial damage, the mechanism underlying early endothelial insult in prediabetes remains obscure. We used a nonobese high-calorie (HC)-fed rat model with hyperinsulinemia, hypercholesterolemia, and delayed development of hyperglycemia to unravel this mechanism. Compared with aortic rings from control rats, HC-fed rat rings displayed attenuated acetylcholine-mediated relaxation. While sensitive to nitric oxide synthase (NOS) inhibition, aortic relaxation in HC-rat tissues was not affected by blocking the inward-rectifier potassium (Kir) channels using BaCl2 Although Kir channel expression was reduced in HC-rat aorta, Kir expression, endothelium-dependent relaxation, and the BaCl2-sensitive component improved in HC rats treated with atorvastatin to reduce serum cholesterol. Remarkably, HC tissues demonstrated increased reactive species (ROS) in smooth muscle cells, which was reversed in rats receiving atorvastatin. In vitro ROS reduction, with superoxide dismutase, improved endothelium-dependent relaxation in HC-rat tissues. Significantly, connexin-43 expression increased in HC aortic tissues, possibly allowing ROS movement into the endothelium and reduction of eNOS activity. In this context, gap junction blockade with 18-ß-glycyrrhetinic acid reduced vascular tone in HC rat tissues but not in controls. This reduction was sensitive to NOS inhibition and SOD treatment, possibly as an outcome of reduced ROS influence, and emerged in BaCl2-treated control tissues. In conclusion, our results suggest that early metabolic challenge leads to reduced Kir-mediated endothelium-dependent hyperpolarization, increased vascular ROS potentially impairing NO synthesis and highlight these channels as a possible target for early intervention with vascular dysfunction in metabolic disease. SIGNIFICANCE STATEMENT: The present study examines early endothelial dysfunction in metabolic disease. Our results suggest that reduced inward-rectifier potassium channel function underlies a defective endothelium-mediated relaxation possibly through alteration of nitric oxide synthase activity. This study provides a possible mechanism for the augmentation of relatively small changes in one endothelium-mediated relaxation pathway to affect overall endothelial response and highlights the potential role of inward-rectifier potassium channel function as a therapeutic target to treat vascular dysfunction early in the course of metabolic disease.

The HTLV-1 oncoprotein Tax is modified by the ubiquitin related modifier 1 (Urm1).

BACKGROUND: Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy secondary to chronic human T-cell lymphotropic virus 1 infection, triggered by the virally encoded oncoprotein Tax. The transforming activity and subcellular localization of Tax is strongly influenced by posttranslational modifications, among which ubiquitylation and SUMOylation have been identified as key regulators of the nuclear/cytoplasmic shuttling of Tax, as well as its ability to activate NF-κB signaling. RESULTS: Adding to the complex posttranslational modification landscape of Tax, we here demonstrate that Tax also interacts with the ubiquitin-related modifier 1 (Urm1). Conjugation of Urm1 to Tax results in a redistribution of Tax to the cytoplasm and major increase in the transcription of the NF-ĸB targets Rantes and interleukin-6. Utilizing a tax-transgenic Drosophila model, we show that the Urm1-dependent subcellular targeting of Tax is evolutionary conserved, and that the presence of Urm1 is strongly correlated with the transcriptional output of Diptericin, an antimicrobial peptide and established downstream target of NF-κB in flies. CONCLUSIONS: These data put forward Urm1 as a novel Tax modifier that modulates its oncogenic activity and hence represents a potential novel target for developing new strategies for treating ATL.

Ki-67 expression predicts biochemical recurrence after radical prostatectomy in the setting of positive surgical margins.

BACKGROUND: Positive surgical margin (PSM) is a predictor of biochemical recurrence (BCR) following radical prostatectomy (RP). Attempts to stratify PSM based on linear length, Gleason score, location and number have failed to add to predictive models using margin status alone. We evaluated the prognostic significance of Ki-67 expression in this setting. METHODS: Immunohistochemical staining for Ki-67 was done on prostatectomy specimens from 117 patients who had a PSM. Ki67 expression was measured at the margin and in the index lesion. Patients were dichotomized based on Ki-67 expression into three groups. Group 1 with no Ki-67 expression, Group 2 with Ki-67 ≤ 2%, and Group 3 with Ki-67 ≥ 3%. To eliminate the impact of the adjuvant treatment (AT) on the outcome, data were analyzed by the Cox proportional hazards in which AT was Considered as a time-dependent covariate. RESULTS: The discordance rate of Ki-67 expression between matched index lesion and margin specimens was 44/117 (37.6%). There was a trend for higher risk of BCR (HR:2.06, (0.97-4.43), P = 0.06) in patients expressing high Ki67 at the surgical margin although this was not statistically significant. However High Ki-67 expression in the index lesion was an independent predictive factor for BCR in this subset of patients. (HR:4, (1.64-9.80), P = 0.002). CONCLUSION: High Ki67 expression in the index prostate cancer lesion is an independent predictor of BCR in patients with positive surgical margin following radical prostatectomy. Our findings need to be validated in a larger cohort.

The HSP90 Family: Structure, Regulation, Function, and Implications in Health and Disease.

The mammalian HSP90 family of proteins is a cluster of highly conserved molecules that are involved in myriad cellular processes. Their distribution in various cellular compartments underlines their essential roles in cellular homeostasis. HSP90 and its co-chaperones orchestrate crucial physiological processes such as cell survival, cell cycle control, hormone signaling, and apoptosis. Conversely, HSP90, and its secreted forms, contribute to the development and progress of serious pathologies, including cancer and neurodegenerative diseases. Therefore, targeting HSP90 is an attractive strategy for the treatment of neoplasms and other diseases. This manuscript will review the general structure, regulation and function of HSP90 family and their potential role in pathophysiology.

SLC35B4, an Inhibitor of Gluconeogenesis, Responds to Glucose Stimulation and Downregulates Hsp60 among Other Proteins in HepG2 Liver Cell Lines.

SLC35B4, solute receptor for UDP-N-acetylglucosamine and UDP-xylose, is associated with diabetes and predisposing conditions. This study investigated the localization of SLC35B4 and compared the differential expression between a knockdown of SLC35B4 and controls in HepG2. Responsiveness to glucose, expression, and localization were assayed using Western blot and immunostaining. Localization was confirmed using a proximity ligation assay. Two-dimensional (2D) gel electrophoresis and MALDI-TOF were used to identify differentially expressed proteins and pathway analysis was performed. SLC35B4 was increased by 60% upon glucose stimulation and localized in Golgi apparatus and endoplasmic reticulum. Presence of SLC35B4 in the Golgi apparatus suggests its involvement in the biosynthesis of glycoconjugate proteins. Four proteins were markedly under-expressed (Hsp60, HspA8, TUBA1A, and ENO1) and linked to the pathogenesis of diabetes or post-translationally modified by O-GlcNAc. Glucose levels activate SLC35B4 expression. This triggers a downstream effect via Hsp60 and other proteins. We hypothesize that the downstream effect on the proteins is mediated via altering the glycosylation pattern inside liver cells. The downstream cascade ultimately alters the ability of cultured liver cells to inhibit endogenous glucose production, and this could play a role in the association of the above-listed genes with the pathogenesis of diabetes.

ATL-derived exosomes modulate mesenchymal stem cells: potential role in leukemia progression.

BACKGROUND: Exosomes are membrane nano-vesicles secreted by a multitude of cells that harbor biological constituents such as proteins, lipids, mRNA and microRNA. Exosomes can potentially transfer their cargo to other cells, implicating them in many patho-physiological processes. Mesenchymal stem cells (MSCs), residents of the bone marrow and metastatic niches, potentially interact with cancer cells and/or their derived exosomes. In this study, we investigated whether exosomes derived from adult T-cell leukemia/lymphoma (ATL) cells act as intercellular messengers delivering leukemia-related genes that modulate the properties of human MSCs in favor of leukemia. We hypothesized that the cargo of ATL-derived exosomes is transferred to MSCs and alter their functional behavior to support the establishment of the appropriate microenvironment for leukemia. RESULTS: We showed that both ATL cells (C81 and HuT-102) and patient-derived cells released Tax-containing exosomes. The cargo of HuT-102-derived exosomes consisted of miR-21, miR-155 and vascular endothelial growth factor. We demonstrated that HuT-102-derived exosomes not only deliver Tax to recipient MSCs, but also induce NF-κB activation leading to a change in cellular morphology, increase in proliferation and the induction of gene expression of migration and angiogenic markers. CONCLUSIONS: This study demonstrates that ATL-derived exosomes deliver Tax and other leukemia-related genes to MSCs and alter their properties to presumably create a more conducive milieu for leukemia. These findings highlight the contribution of leukemia-derived exosomes in cellular transformation and their potential value as biomarkers and targets in therapeutic strategies.

Acute Exposure to Electronic and Combustible Cigarette Aerosols: Effects in an Animal Model and in Human Alveolar Cells.

BACKGROUND: Smoking electronic cigarettes (ECIG) is promoted as a safer alternative to smoking combustible cigarettes. This study investigates the effects of ECIG aerosol and cigarette smoke (CS) in an animal model and in human alveolar cell cultures (A549). METHODS: Mice were divided into Control, ECIG, and CS. Animals were exposed for 6h/d to either lab air, ECIG or CS, for of 3 days. Total particulate matter exposure for the ECIG was set at higher levels compared to CS. Lung injury was determined by: (1) measurement of wet-to-dry ratio; (2) albumin concentration in the bronchoalveolar lavage fluid; (3) transcriptional expression of inflammatory mediators IL-1ß, IL-6, TNF-α; (4) oxidative stress; (5) assessment of cell death; and (6) lung histopathology. Human alveolar cell cultures were treated with various concentrations of ECIG and CS aerosol extracts and the effects on cell proliferation were evaluated. RESULTS: Wet-to-dry ratio was higher in CS when compared to ECIG. Albumin leak in bronchoalveolar lavage fluid was evident in CS but not in ECIG. ECIG exposure was only associated with a significant increase in IL-1ß. In contrast, CS exposure resulted in significant increases in IL-1ß, IL-6, TNF-α expression, and oxidative stress. TUNEL staining demonstrated significant cell death in CS but not in ECIG. At the cellular level, ECIG and CS extracts reduced cell proliferation, however, CS exhibited effects at lower concentrations. CONCLUSION: Despite higher exposure conditions, ECIG exhibited less toxic effects on lungs of experimental animals and on A549 cell cultures when compared to CS.

The regulation of RhoA at focal adhesions by StarD13 is important for astrocytoma cell motility.

Malignant astrocytomas are highly invasive into adjacent and distant regions of the normal brain. Rho GTPases are small monomeric G proteins that play important roles in cytoskeleton rearrangement, cell motility, and tumor invasion. In the present study, we show that the knock down of StarD13, a GTPase activating protein (GAP) for RhoA and Cdc42, inhibits astrocytoma cell migration through modulating focal adhesion dynamics and cell adhesion. This effect is mediated by the resulting constitutive activation of RhoA and the subsequent indirect inhibition of Rac. Using Total Internal Reflection Fluorescence (TIRF)-based Förster Resonance Energy Transfer (FRET), we show that RhoA activity localizes with focal adhesions at the basal surface of astrocytoma cells. Moreover, the knock down of StarD13 inhibits the cycling of RhoA activation at the rear edge of cells, which makes them defective in retracting their tail. This study highlights the importance of the regulation of RhoA activity in focal adhesions of astrocytoma cells and establishes StarD13 as a GAP playing a major role in this process.

Toxicant content, physical properties and biological activity of waterpipe tobacco smoke and its tobacco-free alternatives.

OBJECTIVES: Waterpipe smoking using sweetened, flavoured tobacco products has become a widespread global phenomenon. In this paper, we review chemical, physical and biological properties of waterpipe smoke. DATA SOURCES: Peer-reviewed publications indexed in major databases between 1991 and 2014. Search keywords included a combination of: waterpipe, narghile, hookah, shisha along with names of chemical compounds and classes of compounds, in addition to terms commonly used in cellular biology and aerosol sizing. STUDY SELECTION: The search was limited to articles published in English which reported novel data on waterpipe tobacco smoke (WTS) toxicant content, biological activity or particle size and which met various criteria for analytical rigour including: method specificity and selectivity, precision, accuracy and recovery, linearity, range, and stability. DATA EXTRACTION: Multiple researchers reviewed the reports and collectively agreed on which data were pertinent for inclusion. DATA SYNTHESIS: Waterpipe smoke contains significant concentrations of toxicants thought to cause dependence, heart disease, lung disease and cancer in cigarette smokers, and includes 27 known or suspected carcinogens. Waterpipe smoke is a respirable aerosol that induces cellular responses associated with pulmonary and arterial diseases. Except nicotine, smoke generated using tobacco-free preparations marketed for 'health conscious' users contains the same or greater doses of toxicants, with the same cellular effects as conventional products. Toxicant yield data from the analytical laboratory are consistent with studies of exposure biomarkers in waterpipe users. CONCLUSIONS: A sufficient evidence base exists to support public health interventions that highlight the fact that WTS presents a serious inhalation hazard.

The quinoxaline di-N-oxide DCQ blocks breast cancer metastasis in vitro and in vivo by targeting the hypoxia inducible factor-1 pathway.

BACKGROUND: Although tumor hypoxia poses challenges against conventional cancer treatments, it provides a therapeutic target for hypoxia-activated drugs. Here, we studied the effect of the hypoxia-activated synthetic quinoxaline di-N-oxide DCQ against breast cancer metastasis and identified the underlying mechanisms. METHODS: The human breast cancer cell lines MCF-7 (p53 wildtype) and MDA-MB-231 (p53 mutant) were treated with DCQ under normoxia or hypoxia. Drug toxicity on non-cancerous MCF-10A breast cells was also determined. In vitro cellular responses were investigated by flow cytometry, transfection, western blotting, ELISA and migration assays. The anti-metastatic effect of DCQ was validated in the MDA-MB-231 xenograft mouse model. RESULTS: DCQ selectively induced apoptosis in both human breast cancer cells preferentially under hypoxia without affecting the viability of non-cancerous MCF-10A. Cancer cell death was associated with an increase in reactive oxygen species (ROS) independently of p53 and was inhibited by antioxidants. DCQ-induced ROS was associated with DNA damage, the downregulation of hypoxia inducible factor-1 alpha (HIF-1α), and inhibition of vascular endothelial growth factor (VEGF) secretion. In MCF-7, HIF-1α inhibition was partially via p53-activation and was accompanied by a decrease in p-mTOR protein, suggesting interference with HIF-1α translation. In MDA-MB-231, DCQ reduced HIF-1α through proteasomal-dependent degradation mechanisms. HIF-1α inhibition by DCQ blocked VEGF secretion and invasion in MCF-7 and led to the inhibition of TWIST in MDA-MB-231. Consistently, DCQ exhibited robust antitumor activity in MDA-MB-231 breast cancer mouse xenografts, enhanced animal survival, and reduced metastatic dissemination to lungs and liver. CONCLUSION: DCQ is the first hypoxia-activated drug showing anti-metastatic effects against breast cancer, suggesting its potential use for breast cancer therapy.

Comparison of tobacco-containing and tobacco-free waterpipe products: effects on human alveolar cells.

INTRODUCTION: In recent years, a class of products marketed as "tobacco-free" alternatives for the "health conscious user" has become widely available for waterpipe (hookah, narghile, or shisha) smoking. Their adoption may be in part driven by regulations banning tobacco smoking in public places and by an increasing awareness of the hazards of waterpipe tobacco smoking. Although these products are presented in advertising as a "healthier" choice, very little is known about their health effects. METHODS: In this study, we compared the effects of smoke generated with tobacco-free and conventional tobacco-derived products on human alveolar cells. Smoke was generated with a smoking machine that precisely mimicked the puffing behavior of 15 experienced waterpipe smokers when they used conventional waterpipe tobacco products of their choice and flavor-matched tobacco-free products. Human alveolar epithelial cells (A549) were treated with particulate matter sampled from the smoke, and the effects on cell cycle, proliferation, and doubling time were measured during the subsequent 72hr. RESULTS: We found that smoke from both types of waterpipe products markedly reduced cell proliferation, caused cell cycle arrest at G0/G1, and increased cell doubling time. There were no significant differences across product in any measure. CONCLUSION: Tobacco-free and tobacco-based waterpipe products exert substantial and similar deleterious effects on human lung cells. This study adds to the nascent evidence base indicating that except for exposure to nicotine and its derivatives, use of tobacco-free waterpipe products does not present a reduced health risk relative to the use of conventional tobacco-based products.