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.

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.

JC virus (JCV) is the aetiological agent of progressive multifocal leukoencephalopathy (PML), a fatal, demyelinating disease of the brain affecting people with AIDS. Although immunosuppression is involved in infection of the brain by JCV, a direct influence of human immunodeficiency virus type 1 (HIV-1) has also been established. The Tat protein of HIV-1 has been implicated in activation of the cytokine transforming growth factor (TGF)-β in HIV-1-infected cells and in stimulating JCV gene transcription and DNA replication in oligodendroglia, the primary central nervous system cell type infected by JCV in PML. This study demonstrated that Tat can cooperate with SMAD proteins, the intracellular effectors of TGF-β, at the JCV DNA control region (CR) to stimulate JCV gene transcription. Tat stimulated JCV early gene transcription in KG-1 oligodendroglial cells when expressed via transfection or added exogenously. Using chromatin immunoprecipitation, it was shown that exogenous Tat enhanced binding of SMAD2, -3 and -4 and their binding partner Fast1 to the JCV CR in living cells. When SMAD2, -3 and -4 were expressed together, Tat, expressed from plasmid pTat, stimulated transcription from both early and late gene promoters, with the early promoter exhibiting stimulation of >100-fold. Tat, SMAD4 and JCV large T-antigen were all visualized in oligodendroglial cells at the border of an active PML lesion in the cerebral frontal lobe. These results revealed a positive reinforcement system in which the SMAD mediators of the TGF-β system act cooperatively with Tat to stimulate JCV gene transcription.

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.

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. © 2016 Wiley Periodicals, Inc.