Chemoresistance of cancer floating cells is independent of their ability to form 3D structures: Implications for anticancer drug screening.

Three-dimensional (3D) culture systems such as floating spheroids (FSs) and floating tumorspheres (FTs) are widely used as tumor models of chemoresistance. FTs are considered to be enriched in cancer stem-like cells (CS-LCs). In this study, we used cancer cell lines (lung H460, prostate LnCAP, and breast MCF-7) able to form FSs under anchorage-independent conditions and compared with cell lines (prostate PC3 and breast MDA-MB-231) that cannot form FSs under similar conditions. Independent of their ability to form FTs all cell lines growing under anchorage-independent conditions become highly resistant to obatoclax, colchicine, and hydroxyurea. We used anti-E-cadherin antibody (that blocked the formation of FSs) and demonstrated that floating LnCAP cells showed similar chemoresistance regardless of the formation of spheroids. Our results demonstrate that the development of chemoresistance is not because of the formation of a complex 3D structure and/or enrichment of CS-LCs but is likely the result of cell detachment per se and their ability to survive under anchorage-independent conditions. We propose that FSs and FTs could be useful models to study chemoresistance of cancer cells associated with cell detachment (e.g., circulating tumor cells) but they may not be representative of other types of chemoresistance that arise in vivo in attached cells.

MnTBAP Inhibits Bleomycin-Induced Pulmonary Fibrosis by Regulating VEGF and Wnt Signaling.

Cellular oxidative stress is implicated not only in lung injury but also in contributing to the development of pulmonary fibrosis. We demonstrate that a cell-permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger, manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) significantly inhibited bleomycin-induced fibrogenic effects both in vitro and in vivo. Further investigation into the underlying mechanisms revealed that MnTBAP targets canonical Wnt and non-canonical Wnt/Ca2+ signaling pathways, both of which were upregulated by bleomycin treatment. The effect of MnTBAP on canonical Wnt signaling was significant in vivo but inconclusive in vitro and the non-canonical Wnt/Ca2+ signaling pathway was observed to be the predominant pathway regulated by MnTBAP in bleomycin-induced pulmonary fibrosis. Furthermore, we show that the inhibitory effects of MnTBAP involve regulation of VEGF which is upstream of the Wnt signaling pathway. Overall, the data show that the superoxide scavenger MnTBAP attenuates bleomycin-induced pulmonary fibrosis by targeting VEGF and Wnt signaling pathways. J. Cell. Physiol. 232: 506-516, 2017. © 2016 Wiley Periodicals, Inc.

Anti-tumorigenic effects of a novel digitoxin derivative on both estrogen receptor-positive and triple-negative breast cancer cells.

While there are targeted treatments for triple positive breast cancers, lack of specific biomarkers for triple-negative breast cancers (TNBC) has hindered the development of therapies for this subset of cancers. In this study, we evaluated the anticancer properties of cardiac glycoside Digitoxin (Dtx) and its synthetic analog MonoD on breast cancer cell lines MCF-7 (estrogen receptor-positive breast cancer) and MDA-MB-468 (triple-negative breast cancer). Both cardiac glycosides, at concentrations within the therapeutic range, increased the fraction of cells in the G0/G1 phase of the cell cycle, decreased viability, and inhibited the migration of MCF-7 and MDA-MB-468 cells. Both cardiac glycosides increased production of superoxide and induced apoptosis in both cell types. Reduced protein levels of nuclear factor kappa B and IkappaB kinase-beta were found in cardiac glycoside-treated cells, indicating that the cellular effects of these compounds are mediated via nuclear factor kappa B pathway. This study demonstrates the cytotoxic potential of digitoxin, and more importantly its synthetic analog MonoD, in the treatment of triple-positive breast cancer and more importantly the aggressive triple-negative breast cancer. Collectively, this study provides a basis for the reevaluation of cardiac glycosides in the treatment of breast cancer and more importantly reveals their potential in the treatment of triple-negative breast cancers.

Effects of titanium dioxide nanoparticles on human keratinocytes.

Titanium dioxide (TiO2) is a ubiquitous whitening compound widely used in topical products such as sunscreens, lotions and facial creams. The damaging health effects of TiO2 inhalation has been widely studied in rats, mice and humans showing oxidative stress increase, DNA damage, cell death and inflammatory gene upregulation in lung and throat cells; however, the effects on skin cells from long-term topical use of various products remain largely unknown. In this study, we assessed the effect of specific TiO2 nanoparticles (H2TiO7) on a human keratinocyte cell line (HaCaT). We performed a comparative analysis using three TiO2 particles varying in size (Fine, Ultrafine and H2TiO7) and analyzed their effects on HaCaTs. There is a clear dose-dependent increase in superoxide production, caspase 8 and 9 activity, and apoptosis in HaCaTs after treatment with all three forms of TiO2; however, there is no consistent effect on cell viability and proliferation with either of these TiO2 particles. While there is data suggesting UV exposure can enhance the carcinogenic effects of TiO2, we did not observe any significant effect of UV-C exposure combined with TiO2 treatment on HaCaTs. Furthermore, TiO2-treated cells showed minimal effects on VEGF upregulation and Wnt signaling pathway thereby showing no potential effect on angiogenesis and malignant transformation. Overall, we report here an increase in apoptosis, which may be caspase 8/Fas-dependent, and that the H2TiO7 nanoparticles, despite their smaller particle size, had no significant enhanced effect on HaCaT cells as compared to Fine and Ultrafine forms of TiO2.

A proteomics approach to identifying key protein targets involved in VEGF inhibitor mediated attenuation of bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a life expectancy of less than 5 years post diagnosis for most patients. Poor molecular characterization of IPF has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies. In this study, we have integrated a label-free LC-MS based approach with systems biology to identify signaling pathways and regulatory nodes within protein interaction networks that govern phenotypic changes that may lead to IPF. Ingenuity Pathway Analysis of proteins modulated in response to bleomycin treatment identified PI3K/Akt and Wnt signaling as the most significant profibrotic pathways. Similar analysis of proteins modulated in response to vascular endothelial growth factor (VEGF) inhibitor (CBO-P11) treatment identified natural killer cell signaling and PTEN signaling as the most significant antifibrotic pathways. Mechanistic/mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) were identified to be key mediators of pro- and antifibrotic response, where bleomycin (BLM) treatment resulted in increased expression and VEGF inhibitor treatment attenuated expression of mTOR and ERK. Using a BLM mouse model of pulmonary fibrosis and VEGF inhibitor CBO-P11 as a therapeutic measure, we identified a comprehensive set of signaling pathways and proteins that contribute to the pathogenesis of pulmonary fibrosis that can be targeted for therapy against this fatal disease.

Simvastatin pretreatment prevents ambient particle-induced lung injury in mice.

Air particulate pollution negatively affects the health of the population exposed, being the lung the main target organ. Simvastatin (SV) is widely used for the prevention and risk reduction of coronary disease. Its pleiotropic effects may provide benefit for lung diseases. Here, we investigated the preventive effect of simvastatin pretreatment on acute intranasal exposure to ROFA (Residual Oil Fly Ash), and UAP (Urban Air Particle from Buenos Aires). Male BALB/c mice were randomized in two groups to receive either saline (control, C) solution or SV (1 mg/kg bw /day; ip) for 14 days. After SV treatment, ROFA or UAP (1 mg/kg bw) or saline were intranasally delivered for 24 hours generating 4 subgroups for the ROFA experiment (C, SV, ROFA and SV+ROFA) and 3 subgroups for the UAP experiment (C, SV, UAP and SV+UAP). Biomarkers of lung injury were examined in BAL cells evaluating total cell number (TCN), cell differential (CD) and superoxide anion generation (O2-), in lung homogenates assessing superoxide dismutase activity (SOD) and tumor necrosis factor α (TNFα); and in blood samples determining interleukin 6 (IL-6) production. ROFA and UAP produced an acute pulmonary injury, characterized by an increase in BAL, TCN and neutrophilic inflammatory influx, a rise in O2- generation, and production of the proinflammatory TNFα cytokine. SV pretreatment had no significant effect per se on any of these biomarkers but prevented the pulmonary cytotoxicity and inflammation induced by ROFA and UAP. Our results encourage further studies to determine the preventive effects on lung injury induced by air pollutants.

Rationale for the use of sphingosine analogues in COVID-19 patients.

Despite the recent announcement of promising drug candidates to treat COVID-19, there is currently no effective antiviral drug or vaccine. There is strong evidence that acute lung injury/acute respiratory distress syndrome (ALI/ARDS), likely triggered by a cytokine storm, is responsible for the severity of disease seen in COVID-19 patients. In support of this hypothesis, pilot studies using IL-6 receptor inhibitors such as tocilizumab have shown promising results. Therefore, the use of drugs or cocktails of drugs with broader ability to inhibit these cytokine receptors is likely to be effective. In this article, we propose the use of sphingosine analogues, which have been shown to mitigate acute lung damage in animal models of ALI/ARDS, as early adjuvant therapies to prevent and/or mitigate the cytokine response in COVID-19 patients. This proposal is based on the ability of these drugs to decrease the production of IL-6 and other cytokines. The potential application of fingolimod (FTY720), the oldest sphingosine analogue approved for the treatment of multiple sclerosis, in the early stages of COVID-19 is discussed in more detail as a prototype drug.

Low doses of urban air particles from Buenos Aires promote oxidative stress and apoptosis in mice lungs.

UNLABELLED: Air pollution consists of a wide range of gaseous and particulate pollutants. Exposure to particulate matter (PM) can cause oxidative stress within the lung, which in turn can negatively impact health. The mechanisms by which PM causes oxidative stress include the release of trace metals or organic components from the particle. Previously, we have characterized urban air particles from downtown Buenos Aires (UAP-BA) and, by using in vivo animal studies, found that they are able to generate lung inflammation. PURPOSE: We studied lung responses to low doses of UAP-BA (15 µg), with special emphasis on oxidative balance. METHODS: We assessed cell viability, total cell number (TCN) and cell differential (CD) on bronchoalveolar lavages (BAL), oxidative metabolism in lung homogenates by tertbutylhydroperoxide-initiated chemiluminescence (CL), thiobarbituric reactive substances (TBARS), total reactive antioxidant potential (TRAP), reduced glutathione (GSH), and apoptosis in lung sections. RESULTS: We found that low UAP-BA exposure increases TCN, modifies CD, and decreases cell viability in the BAL. In lung homogenates, TBARS and CL rose while TRAP and GSH showed no alteration when compared to controls. Occurrence of apoptosis evaluated by TUNEL assay was markedly augmented in UAP-BA exposed animals. CONCLUSIONS: Our data further implicate oxidative stress as a possible inducer of apoptosis in lungs from animals exposed to low concentrations of this urban environmental contaminant.

Electrical Impedance Spectroscopy for Monitoring Chemoresistance of Cancer Cells.

Electrical impedance spectroscopy (EIS) is an electrokinetic method that allows for the characterization of intrinsic dielectric properties of cells. EIS has emerged in the last decade as a promising method for the characterization of cancerous cells, providing information on inductance, capacitance, and impedance of cells. The individual cell behavior can be quantified using its characteristic phase angle, amplitude, and frequency measurements obtained by fitting the input frequency-dependent cellular response to a resistor-capacitor circuit model. These electrical properties will provide important information about unique biomarkers related to the behavior of these cancerous cells, especially monitoring their chemoresistivity and sensitivity to chemotherapeutics. There are currently few methods to assess drug resistant cancer cells, and therefore it is difficult to identify and eliminate drug-resistant cancer cells found in static and metastatic tumors. Establishing techniques for the real-time monitoring of changes in cancer cell phenotypes is, therefore, important for understanding cancer cell dynamics and their plastic properties. EIS can be used to monitor these changes. In this review, we will cover the theory behind EIS, other impedance techniques, and how EIS can be used to monitor cell behavior and phenotype changes within cancerous cells.

Anti-Tumorigenic Effects of Resveratrol in Lung Cancer Cells Through Modulation of c-FLIP.

BACKGROUND: Resveratrol has been shown to have antioxidant and anti-proliferative properties in multiple cancer types. Here we demonstrate that H460 lung cancer cells are more susceptible to resveratrol treatment in comparison to human bronchial epithelial Beas-2B cells. Resveratrol decreases cell viability and proliferation, and induces significant apoptosis in H460 cells. The apoptosis observed was accompanied by an increase in hydrogen peroxide (H2O2) production, Bid, PARP and caspase 8 activation, and downregulation of pEGFR, pAkt, c-FLIP and NFkB protein expression. Furthermore, treatment with HH2O2 scavenger catalase significantly inhibited resveratrol-induced c-FLIP downregulation, caspase-8 activation and apoptosis. Overexpression of c-FLIP in H460 cells (FLIP cells) resulted in the inhibition of resveratrol-induced HH2O2 production, and a significant increase in resveratrolinduced apoptosis in comparison to H460 cells. In FLIP cells, catalase treatment did not rescue cells from a decrease in cell viability and apoptosis induction by resveratrol as compared to H460 cells. Resveratrol treatment also led to VEGF downregulation in FLIP cells. Furthermore, inhibition of pEGFR or pAkt using erlotinib and LY294002 respectively, enhanced the negative effect of resveratrol on FLIP cell viability and apoptosis. The reverse was observed when FLIP cells were supplemented with EGF, or transfected with WT-AKT plasmid; resulting in a 20% decrease in resveratrol-induced apoptosis. In addition, transfection with WT-AKT plasmid resulted in the inhibition of pro-apoptotic protein activation, and c-FLIP and pAkt downregulation. CONCLUSION: Overall, resveratrol induced apoptosis in H460 lung cancer cells by specifically targeting pAkt and c-FLIP dowregulation by proteasomal degradation in a EGFR-dependent manner.

Improvement of bovine semen quality by removal of membrane-damaged sperm cells with DNA aptamers and magnetic nanoparticles.

In cattle, cryopreservation of semen and sex-sorting kill up to 50% of spermatozoa and decrease the success of assisted insemination (AI). Therefore, significant efforts are being carried out to improve the quality of semen prior to AI. In this work we used the Cell-SELEX technique to select single strand DNA aptamers able to recognize with high affinity and specificity damaged sperm cells generated by heat-treatment. We first isolated aptamers with a conserved two motifs of 6 nucleotides of length that bind to the membrane of heat-treated spermatozoa. Then, we used synthetic biotin-labeled aptamers containing the conserved motif to recognize membrane-damaged cells and separate them from viable cells by the use of avidin-coated superparamagnetic iron oxide nanoparticles (SPION). This procedure improved the quality of semen by significantly increasing the percentage of healthy sperm cells without affecting the rate of blastocyst cleavage. This technique was successfully applied to both unsorted and sex-sorted sperm suspension.

Menadione : sodium orthovanadate combination eliminates and inhibits migration of detached cancer cells.

Exposure of cancer cells to anticancer agents in cultures induces detachment of cells that are usually considered dead. These drug-induced detached cells (D-IDCs) may represent a clinical problem for chemotherapy since they may survive anoikis, enter the circulation, invade other tissues and resume proliferation, creating a metastasis, especially in tissues where the bioavailability of anticancer agents is not enough to eliminate all cancer cells. In this study we evaluated the antiproliferative effect of menadione : sodium orthovanadate (M : SO) combination on A549 lung cancer cells as well as the ability of M : SO to induce cell detachment. In addition, we followed the fate and chemosensitivity of M : SO-induced detached cells. Using transwell chambers, we found that a fraction of the M : SO-induced detached cells were viable and, furthermore, were able to migrate, re-attach, and resume proliferation when re-incubated in drug-free media. The total elimination of A549 detachment-resistant cells and M : SO-induced detached cells were successfully eliminated by equivalent M : SO concentration (17.5 µM : 17.5 µM). Thus, M : SO prevented cell migration. Similar results were obtained on DBTRG.05MG human glioma cells. Our data guarantee further studies to evaluate the in vivo occurrence of D-IDCs, their implications for invasiveness and metastasis and their sensitivity to anticancer drugs.