Clayton Wright

Nitric Oxide Mediates Bleomycin‐induced Angiogenesis and Pulmonary Fibrosis via Regulation of VEGF

Pulmonary fibrosis is a progressive lung disease hallmarked by increased fibroblast proliferation, amplified levels of extracellular matrix deposition and increased angiogenesis. Although dysregulation of angiogenic mediators has been implicated in pulmonary fibrosis, the specific rate‐limiting angiogenic markers involved and their role in the progression of pulmonary fibrosis remains unclear. We demonstrate that bleomycin treatment induces angiogenesis, and inhibition of the central angiogenic mediator VEGF using anti‐VEGF antibody CBO‐P11 significantly attenuates bleomycin‐induced pulmonary fibrosis in vivo. Bleomycin‐induced nitric oxide (NO) was observed to be the key upstream regulator of VEGF via the PI3k/Akt pathway. VEGF regulated other important angiogenic proteins including PAI‐1 and IL‐8 in response to bleomycin exposure. Inhibition of NO and VEGF activity significantly mitigated bleomycin‐induced angiogenic and fibrogenic responses. NO and VEGF are key mediators of bleomycin‐induced pulmonary fibrosis, and could serve as important targets against this debilitating disease. Overall, our data suggests an important role for angiogenic mediators in the pathogenesis of bleomycin‐induced pulmonary fibrosis. J. Cell. Biochem. 116: 2484–2493, 2015.

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.

Autophagy-Induced Apoptosis in Lung Cancer Cells by a Novel Digitoxin Analog

We have synthesized a novel derivative of Digitoxin, termed “MonoD”, which demonstrates cytotoxic effects in lung cancer cells with much higher potency as compared to Digitoxin. Our data show that within 1 h of MonoD treatment, H460 cells showed increased oxidative stress, increased formation of autophagic vacuoles, and increased expression of pro‐autophagic markers Beclin‐1 and LC3‐II. Cells pretreated with MnTBAP, a superoxide scavenger not only lowered superoxide production, but also had lower levels of LC3‐II and Beclin‐1. Prolonged treatment with MonoD‐induced apoptosis in lung cancer cells. We investigated MonoD‐dependent regulation of Akt and Bcl2, proteins that are known regulators of both autophagy and apoptosis. Molecular and pharmacologic inhibitors of Bcl2 and Akt, when combined with MonoD, led to higher expression of LC3‐II and Beclin‐1 as compared to MonoD alone, suggesting a repressive effect for these proteins in MonoD‐dependent autophagy. Pretreatment of cells with an autophagy inhibitor repressed the apoptotic potential of MonoD, confirming that early autophagic flux is important to drive apoptosis. Therapeutic entities such as MonoD that target multiple pathways such as autophagy and apoptosis may prove advantageous over current therapies that have unimodal basis for action and may drive sustained tumor regression, which is highly desirable. J. Cell. Physiol. 231: 817–828, 2016.

Anti-Tumor Effects of Cardiac Glycosides on Human Lung Cancer Cells and Lung Tumorspheres: ANTI-TUMOR EFFECTS OF CARDIAC GLYCOSIDES

Lung cancer is a leading cause of cancer‐related death in the United States. Although several drugs have been developed that target individual biomarkers, their success has been limited due to intrinsic or acquired resistance for the specific targets of such drugs. A more effective approach is to target multiple pathways that dictate cancer progression. Cardiac glycosides demonstrate such multimodal effects on cancer cell survival, and our aim was to evaluate the effect of two naturally occurring monosaccaridic cardiac glycosides—Convallatoxin and Peruvoside on lung cancer cells. Although both drugs had significant anti‐proliferative effects on H460 and Calu‐3 lung cancer cells, Convallatoxin demonstrated twofold higher activity as compared to Peruvoside using both viability and colony forming assays, suggesting a role for the aglycone region in dictating drug potency. The tumor suppressor p53 was found to be important for action of both drugs—p53‐underexpressing cells were less sensitive as compared to p53‐positive H460 cells. Further, assessment of p53‐underexpressing H460 cells showed that drugs were able to arrest cells in the G0/G1 phase of the cell cycle in a dose‐dependent manner. Both drugs significantly inhibited migration and invasion of cancer cells and decreased the viability of floating tumorspheres. An assessment of intracellular pathways indicated that both drugs were able to modulate proteins that are involved in apoptosis, autophagy, cell cycle, proliferation, and EMT. Our data suggest, a promising role for cardiac glycosides in lung cancer treatment, and provides impetus for further investigation of the anti‐cancer potential of this class of drugs. J. Cell. Physiol. 232: 2497–2507, 2017.

Formation of Tumorspheres with Increased Stemness without External Mitogens in a Lung Cancer Model

Like with most solid tumors, the presence of a subpopulation of cancer stem cells (CSCs) or cancer stem-like cells (CS-LCs) has been associated with chemoresistance and tumor relapse in lung cancer cells. In the absence of serum, CSCs/CS-LCs have the ability to grow as lung tumorspheres (LTSs), and this system is routinely used for isolation and characterization of putative CSCs/CS-LCs. Methods to isolate LTSs are usually performed in serum-free media supplemented with specific additives such as epidermal growth factor and basic fibroblast growth factor. In this study, we report the generation of LTSs without the addition of any external mitogenic stimulation. LTSs generated in this manner demonstrated several traits usually associated with increased stemness such as elevated expression of the stemness-associated marker Sox2 and increased chemoresistance to conventional anticancer drugs. In addition, we report that the FDA-approved drug Digitoxin, at concentration close to its therapeutic level, decreased the viability of LTSs and downregulated Sox2 independent of the PI3K/AKT pathway. The potential use of LTSs generated without the addition of any external mitogenic stimulation to study the role of specific factor(s) associated with stemness properties is also discussed.

Digitoxin and its synthetic analog MonoD have potent antiproliferative effects on lung cancer cells and potentiate the effects of hydroxyurea and paclitaxel

Despite significant advances in the understanding of lung cancer biology, the prognosis of cancer patients remains poor. Part of the failure of anticancer therapy is due to intratumoral heterogeneity in these patients that limits the efficacy of single agents. Therefore, there is an urgent need for new anticancer drugs or drug combination regimens that possess increased activity against all cellular subtypes found within the tumor. In this study, we evaluated the in vitro antiproliferative activity of the cardiac glycosides (CGs) digitoxin and its synthetic analog MonoD on H460 lung cancer cells grown under different culture conditions. The CGs were tested alone in H460 cells under routine culture as well as in cells growing under short (24–72 h) and prolonged serum starvation (7 days) in order to evaluate the activity of drugs on cancer cells under varied degrees of proliferation. Our results showed that both CGs, and MonoD in particular, have potent antiproliferative activity at clinically relevant concentrations against cells in all the tested culture conditions. In contrast, paclitaxel, hydroxyurea and colchicine were only active in cells growing in routine culture conditions, and relatively inactive in serum-starved conditions. Importantly, both CGs were able to potentiate the effect of clinically relevant concentrations of hydroxyurea or paclitaxel in serum-starved conditions. When paclitaxel was used in combination with CGs, the highest antiproliferative effect was obtained when paclitaxel was administered first, followed by either digitoxin or MonoD. Our results indicate that CGs have potential clinical applications in translational oncology especially in combination with other drugs, and warrants further investigation of CGs in more advanced preclinical models of lung cancer.

S-Nitrosylation of Bcl-2 Negatively Affects Autophagy in Lung Epithelial Cells†

Autophagy is a catabolic cellular mechanism involving lysosomal degradation of unwanted cellular components. Interaction between Beclin‐1 and Bcl‐2 proteins is known to play a critical role in the initiation of autophagy. We report that malignantly transformed lung epithelial cells are resistant to autophagy and express lower basal levels of autophagic proteins, Beclin‐1 and LC3‐II as compared to non‐tumorigenic cells. Additionally, increased levels of nitric oxide (NO) and Bcl‐2 were observed in transformed cells. Nitric oxide was found to negatively regulate autophagy initiation and autophagic flux by nitrosylating Bcl‐2 and stabilizing its interaction with Beclin‐1, resulting in inhibition of Beclin‐1 activity. An increase in the apoptotic initiator caspase‐9 and the apoptosis and autophagy‐associated kinase p38/MAPK in both cell types indicated possible autophagy–apoptosis crosstalk. Pre‐treatments with ABT‐737 (Bcl‐2 inhibitor) and aminoguanidine (NO inhibitor), and transfection with a non‐nitrosylable Bcl‐2 cysteine double‐mutant plasmid resulted in increased autophagic flux (LC3‐II/p62 upregulation) corresponding with decreased S‐nitrocysteine expression, thus corroborating the regulatory role of Bcl‐2 S‐nitrosylation in autophagy. In conclusion, our study reveals a novel mechanism of autophagy resistance via post‐translational modification of Bcl‐2 protein by NO, which may be critical in driving cellular tumorigenesis. J. Cell. Biochem. 117: 521–532, 2016.

Anti-Tumorigenic Potential of a Novel Orlistat-AICAR Combination in Prostate Cancer Cells

Prostate cancer (PCa) is one of the leading causes of cancer‐related deaths in men worldwide. Fatty acid synthase (FASN) is reported to be overexpressed in several cancers including PCa, and this has led to clinical cancer treatments that utilize various FASN inhibitors such as the anti‐obesity drug, Orlistat. However, pharmacological limitations have impeded the progress in cancer treatments expected thus far with FASN inhibition. In this study, we investigated a novel therapeutic combination to enhance the toxic potential of Orlistat in three different PCa cell‐lines (DU145, PC3, and LNCaP). We show that Orlistat and 5‐Aminoimidazole‐4‐carboxamide ribonucleotide (AICAR) (AMP‐activated protein kinase [AMPK] activator) co‐treatment induces significant downregulation of two key fatty acid synthesis regulatory proteins (FASN, Sterol regulatory element‐binding protein 1 [SREBP‐1c]) as compared to control and Orlistat alone. Orlistat and AICAR co‐treatment induced a significant decrease in cell viability and proliferation, and a significant increase in apoptosis in all three PCa cell‐lines. Apoptosis induction was preceded by a marked increase in reactive oxygen species (ROS) production followed by G0/G1 cell cycle arrest and activation of pro‐apoptotic caspases. We also observed a significant decrease in migration potential and VEGF expression in Orlistat and AICAR co‐treated samples in all three PCa cell‐lines. Compound C (AMPK inhibitor) negatively affected some of the enhanced anti‐cancer effects observed with Orlistat treatment. We conclude that AICAR co‐treatment potentiates the anti‐proliferative effects of Orlistat at a low dose (100 µM), and this combination has the potential to be a viable and effective therapeutic option in PCa treatment. J. Cell. Biochem. 118: 3834–3845, 2017.

Effects of titanium dioxide nanoparticles on human keratinocytes

Abstract 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.

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.